Bull. Org. mond. Sante 1963, 29, 565-578Bull. Wld Hlth Org. i

M ycobacteria: Laboratory Methods for Testing DrugSensitivity and Resistance

G. CANETTI,1 S. FROMAN,2 J. GROSSET,3 P. HAUDUROY,4 MILOSLAVA LANGEROVA,5H. T. MAHLER,6 GERTRUD MEISSNER,7 D. A. MITCHISON8 & L. gULA 9

In its seventh report, published in 1960, the WHO Expert Committee on Tuberculosis"noted the need for international standards for the definition and determination of drugresistance which will permit comparisons to be made from one area to another, and recom-mended that the World Health Organization take appropriate steps to establish suchstandards ".10 Acting on this recommendation, WHO took the first step towards standard-ization by convening in Geneva, in December 1961, an informal international meeting ofspecialists in the bacteriology of tuberculosis. At this meeting an attempt was made toformulate prerequisites for reliable sensitivity tests and to specify the technical proceduresfor them.

The first part of the present paper is a joint contribution by the participants in themeeting, summarizing the general conclusions reached and recommendations made withregard to tests ofsensitivity to the three main antituberculosis drugs-isoniazid, streptomycinand p-aminosalicylic acid. The other three parts describe, in turn, three different tests fordetermining drug sensitivity-the absolute-concentration method, the resistance-ratiomethod and the proportion method-that are generally considered to give reasonablyaccurate results.

I. INTRODUCTION"1

Since the introduction of potent antimicrobialdrugs, great advances have been made in the controlof tuberculosis and other infectious diseases.However, with few exceptions, the activity of thesedrugs has been limited by the appearance of strainsof bacteria which are capable of growth in the pre-

sence of unusually high concentrations of the drugs.Tubercle bacilli have been found that are resistantto each of the drugs introduced for the treatment oftuberculosis. Resistant strains can be produced fromcultures in the laboratory and have also beenobtained from experimental animals and from

I Chef de Laboratoire, Service de la Tuberculose, InstitutPasteur, 25 rue du Docteur Roux, Paris 15e, France.

I Director, Microbiological Research Laboratory, OliveView Hospital, Olive View, Calif., USA.

' Assistant, Institut Pasteur, 25 rue du Docteur Roux,Paris 15e, France.

' Directeur, Institut de Bacteriologie, d'Hygiene et deVirologie, Facult6 de Medecine, 19 avenue Cesar-Roux,Lausanne, Switzerland.

' Research Assistant, Tuberculosis Research Institute,Srob,irova 48, Prague 10, Czechoslovakia.

' Chief Medical Officer, Tuberculosis, Division ofCommunicable Diseases, World Health Organization,Geneva, Switzerland.

' Tuberculosis Research Institute, Borstel uiber BadOldesloe, Germany.

8 Director, Unit for Research on Drug Sensitivity inTuberculosis (Medical Research Council of Great Britain),Postgraduate Medical School of London, Ducane Road,London W.12, England.

I Chief, Department of Bacteriology and Epidemiology,Tuberculosis Research Institute, Srobirova 48, Prague 10,Czechoslovakia. Formerly Medical Officer, Tuberculosis,Division of Communicable Diseases, World Health Organi-zation, Geneva, Switzerland.

Wld Hlth Org. techn. Rep. Ser., 1960, 195, 11.11 Prepared by the participants in an informal meeting

of Advisers on Laboratory Methods for the Drug Sensitivity/Resistance Determination of Mycobacteria, held in Genevafrom 4 to 7 December 1961: Dr G. Canetti, Dr S. Froman,Professor P. Hauduroy, Dr Miloslava LangerovA, Dr H. T.Mahler, Professor Gertrud Meissner, Dr D. A. Mitchisonand Dr L. Sula.

1342 -565-

566 G. CANETTI AND OTHERS

patients treated with the drug concerned. In themajority of instances, these strains arise by theselective growth of small numbers of resistant mutantbacilli present in the bacterial population before itcomes into contact with the drug. In a few instances,tubercle bacilli may be naturally resistant to a drugbefore they have ever come into contact with it.Thus the bovine type may be naturally resistant top-aminosalicylic acid (PAS) and to pyrazinamide.The importance of bacterial resistance has been

reviewed by the WHO Expert Committee on Anti-biotics which, in its second report,1 made thefollowing observation:

" Bacterial resistance to antibiotics is the principalobstacle to their successful therapeutic use. Whenresistance develops during a course of treatment, it maydeprive an antibiotic of its proper therapeutic effect in thepatient being treated. More important in the long runis the effect on the general community, since the elimina-tion of sensitive strains and the dissemination of resistantones leads to a situation in which many infections areresistant ab initio and alternative treatment must beadopted. For this reason, the estimation of bacterialsensitivity or resistance to antibiotics has assumed greatimportance. Such estimations are an essential prerequi-site for the rational use of antibiotics and for preservingthe efficacy of this important group of therapeuticsubstances."

The widespread use of two or more drugs givensimultaneously in treatment has markedly reducedthe emergence of drug resistance during treatment,but, in the event of failure to use such combinedtherapy in the most efficient manner, patients con-tinue to produce large numbers of drug-resistantorganisms in their sputum. Other patients maybecome infected with these resistant bacilli and arethen far less likely to respond satisfactorily to thedrug concerned.A major problem in any control programme of

tuberculosis based on the application of mass chemo-therapy is the danger of the widespread disseminationof drug-resistant tubercle bacilli. There are reasonsto believe that strains resistant to isoniazid andstreptomycin did not occur in the community priorto the introduction of these drugs, and it is probablytrue that there was very little resistance to PAS.Surveys of the prevalence of primary drug resistancein many countries have indicated that from 3% to

1 WHO Expert Committee on Antibiotics (1961) WldHith Org. techn. Rep. Ser., 210, 3.

15% of strains from previously untreated patientsmay be resistant to one or more of the three standarddrugs. It is possible that the prevalence of drug-resistant strains may be increasing, particularly inthose countries where the usual treatment of patientsis unsatisfactory.

Laboratory tests of the sensitivity of tuberclebacilli to chemotherapeutic drugs serve three mainpurposes: first, they can be used as a guidance in thechoice of the first course of chemotherapy to be givento the patient. Secondly, they may be of value inconfirming that drug resistance has emerged when apatient has failed to show a satisfactory bacterio-logical response to treatment, and may guide thechoice of a further course of treatment with differentdrugs. Thirdly, they may be employed to estimatethe prevalence of primary and of acquired drugresistance in a community. For each of thesepurposes, it is of great importance to use a reliabletechnique in performing the test. Unfortunately,many different techniques and methods of inter-preting the results are in use (Rist & Crofton 2). It isprobable that many of these techniques fail todistinguish with any precision between sensitive andresistant organisms.

Before prerequisites for reliable sensitivity testscan be formulated and technical procedures for themlaid down, agreement must be reached on thedefinition of the terms " sensitive " and " resistant ".In our opinion, " sensitive " and " resistant " strainsof Mycobacterium tuberculosis may be defined asfollows: " Sensitive" strains are those that havenever been exposed to the main antituberculosisdrugs (" wild " strains) and that respond to thesedrugs, generally in a remarkably uniform manner." Resistant " strains are those that differ from sen-sitive strains in their capacity to grow in the presenceof higher concentrations of a drug. This definitionof resistance is based on the laboratory response;strains that are resistant in this sense do not neces-sarily fail to respond to the usual doses of the drugin the lesions of the patient. However, a diminishedclinical response is likely to occur whenever resistanceis demonstrated in the laboratory, even though theextent or degree of that resistance is small.

In the following discussion of possible methods ofdetermining drug sensitivity, we have borne inmind the need for a test that is accurate, repro-ducible, economical and rapid. Consideration has

2Rist, N. & Crofton, J. (1960) Bull. int. Un. Tuberc.,30, 2.

LABORATORY METHODS FOR TESTING DRUG SENSITIVITY OF MYCOBACTERIA 567

been given only to the three main antituberculosisdrugs-isoniazid, streptomycin and PAS-althoughwe fully realize that accurate tests for resistance tothe minor drugs are also of considerable importance.

STANDARDIZATION OF A SENSITIVITY TEST

Techniques for performing sensitivity tests varywidely in different laboratories, both in the methodsemployed and in the interpretation of the results.It seems probable that some of these techniquesclassify a proportion of wild strains as resistant or,conversely, a proportion of resistant strains assensitive. A satisfactory method should yield aminimum of misclassification in either of thesedirections. For this reason, we recommend that anymethod employed should be standardized in termsof the response of a sample of wild strains. Sincethere is some evidence that strains of Myco. tuber-culosis vary in their sensitivity to chemotherapeuticagents according to the country of their origin, itwould be desirable to obtain these sample strainsfrom one or more countries where the prevalentorganisms have been adequately studied and wherethe therapeutic response of patients to the drugconcerned is known. Standardization using samplesof wild strains may present problems owing todifficulties of storage and transportation of thestrains.' A standard sensitive strain (possiblyH37Rv) may be employed as a sub-standard if itsresponse to the drug concerned has been comparedin one laboratory with a suitable sample of wildstrains by the method to be employed.

TYPES OF SENSMTIVrrY TEST IN CURRENT USE

Sensitivity tests in current use may be classified,in the first place, as direct or indirect. In direct tests,the sputum homogenate or other pathologicalmaterial is cultured directly on drug-containingmedium. The tests are performed only on thosespecimens that contain adequate numbers of acid-fast bacilli as shown by direct smear examination(at least one bacillus in ten high-power microscopicfields), since results may not be reliable when the

1 gula, L., Sundaresan, T. K. & Langerova, M. (1960)Bull. Wld Hlth Org., 23, 635.

growth on culture is scanty. The exact proceduresto be adopted are also determined in most methodsby the number of bacilli seen in direct smearexaminations.

In many laboratories, direct sensitivity tests arenot performed-partly, because it is not consideredpractical to examine smears as a routine beforeinoculation of the culture medium and, partly,because direct sensitivity tests performed by sometechniques are not considered as reliable as thosedone by the indirect method. Whatever method isused, indirect tests will be necessary for cultureswhere the direct smear examination was negativeor only scantily positive.The main reason for the use of direct tests is that

they reduce the time between obtaining the specimenfrom the patient and reading the sensitivity resultfrom about seven weeks, the time necessary for theindirect test, to four weeks. With certain patients-for instance, those with progressive disease about toreceive a re-treatment course of chemotherapy-it isimportant to obtain the results of a reliable test assoon as possible; direct sensitivity tests are ofparticular value in these circumstances.

In indirect tests, the drug-containing medium isinoculated from the primary diagnostic culture.Indirect tests may be classified into three maincategories: (a) the absolute-concentration method;(b) the resistance-ratio method; and (c) the propor-tion method. Examples of method (a) have beendescribed by the United States Veterans Administra-tion 2 as well as by Meissner in Part II of this paper(see page 570). Method (b), which is used in investiga-tions under the auspices of the Medical ResearchCouncil of Great Britain, is described by Mitchisonin Part III of this paper (see page 573) and method (c)is described by Canetti & Grosset in Part IV (seepage 574).Each of these three indirect tests may be adapted

for use as a direct test. In the absolute-concentrationand resistance-ratio methods, additional controlsare necessary; several different inocula of thestandard strain should be employed to correspondin bacillary content to the expected range of positivityof the test material. An advantage of the proportionmethod is that this elaboration is unnecessary sincethe direct and indirect methods are virtually identical.

' United States Veterans Administration, Department ofMedicine and Surgery (1960) Tuberculosis laboratory methodsfor the V. A. Armed Forces Cooperative Study on the Chemo-therapy of Tuberculosis, Washington, D.C., p. 9.

568 G. CANETITI AND OTHERS

RELATIVE MERITS OF THE METHODS

A satisfactory test by the absolute-concentrationmethod is obtained only:

(a) if the inoculum employed is adequatelystandardized in size; and

(b) if the critical concentration of the drug hasbeen standardized for the laboratory and for themethod concerned by reference to an adequatesample of wild strains.

In such a test, it is usually necessary to standardizethe method before it is brought into general use,and control studies must be done at intervals to seethat no alterations in the inoculum or the mediumhave occurred. Without adequate standardizationit has proved particularly unsatisfactory to transferthe findings obtained in one laboratory to anotherlaboratory.

The satisfactory performance of the resistance-ratio method also depends upon adequate standar-dization of the size of the inoculum of both the teststrain and the standard strain, but it is not necessaryto define the critical drug concentration sincea running control is provided by the standardstrain. This method requires slightly more mediumthan the absolute-concentration method. Further-more, the results obtained are very slightly lessaccurate and it is necessary to re-test strains withdoubtful results a little more frequently.

The proportion method has the advantage thatstandardization of the size of the inoculum is notof critical importance since the inoculum size isknown from the viable counts carried out in the test.The results are possibly influenced to a certainextent by the degree of dispersion of the inoculumsuspension, so that it is of particular importance toadhere to the exact procedure for preparing thesuspension. It is also necessary to determine thecorrect critical concentration to be used in the testin the same manner as for the absolute-concentrationmethod. Although no comparison of this and theother two methods has been published, the propor-tion method may perhaps be slightly more accuratethan the others since adjustment is made for animportant source of variation in the results-thesize of inoculum. The test takes slightly longer tocarry out than the other methods, and requires theperformance of quantitative techniques.

RECOMMENDATIONS

We consider that the best .type of sensitivity testis a fully quantitative determination in which theproportion of organisms capable of growth onmedium containing a wide range of drug concen-trations is known. This type of test would provide fullinformation both on the degree of the resistance andon the proportion of resistant organisms at each drugconcentration. However, since such a test requireslarge amounts of medium and is time-consuming, itcannot be recommended as a routine procedure.It will be appreciated that each of the foregoingmethods is a compromise in which one or other ofthe assessments of the results of a test is given thegreatest prominence. A fully quantitative test shouldbe employed whenever the circumstances permit.We are unable to recommend a single standard

method for adoption, but are agreed on the followingtechnical features of sensitivity tests:

1. The medium to be employed should be L6wen-stein-Jensen without potato starch, described as themedium of the International Union against Tuber-culosis (IUT medium).' L6wenstein-Jensen mediumis widely used. The omission of potato starch doesnot appear to influence the results of sensitivity testsand the IUT medium is simpler to prepare.

2. Solutions of the chemotherapeutic drugs can bedissolved in sterile distilled water and added to themedium without further sterilization. However, ifstock solutions of the drugs are to be stored, theyshould be sterile, either as a result of preparing themfrom sterile ampoules of the drug or by filtrationthrough a membrane, sintered-glass or candle filter,but not by Seitz filtration.

3. In reporting the concentrations of drug in themedium (particularly streptomycin or dihydro-streptomycin), the concentration should be thatpresent in the medium before inspissation. Noaccount should be taken of possible inactivationduring inspissation or storage.

4. Drug-containing medium can be stored at 4°Cfor a period not exceeding three months. Suchmedium should not be stored at room temperaturebecause of appreciable destruction of certain drugs.

5. Attention is again drawn to the fundamentalimportance of standardizing all methods of per-forming sensitivity tests. It would be particularlydesirable to achieve standardization (a) in recording

1 Jensen, K. A. (1955) Bull. int. Un. Tuberc., 25, 89.

LABORATORY METHODS FOR TESTING DRUG SENSITIVIY OF MYCOBACTERIA 569

the results in terms of the response of wild strainsof tubercle bacilli, and (b) in respect of the inoculum.

Simple testfor isoniazid resistance

We recommend the following simple test forisoniazid resistance:A representative loopful of the growth on the

primary diagnostic culture is taken with a loop of3-mm external diameter made from thick nichromewire (for example, 22 SWG 1). The amount ofthis growth is about 2 mg (moist weight) visualizedas 2 mm3 on the loop. The loopful of growth isadded to a screw-capped bottle or similar containerwith a volume of approximately 25 ml (for instance,a 1-ounce bottle) containing 12 glass beads (2-3 mmin diameter) and 0.5 ml of sterile distilled water.The bottle is shaken for 10 to 20 seconds by hand andto it is added 9.5 ml of sterile distilled water. As asafety precaution, a fresh cap is put on the bottle,which is then briefly shaken again. From thesuspension so prepared a loopful is taken with a loopof 3-mm external diameter made from thin nichromewire (for example, 27 SWG) and placed on each ofthree slopes of Lowenstein-Jensen medium withoutpotato starch (IUT medium) containing, respectively,0, 0.2 and 1.0 ,ug/ml of isoniazid. The slopes areincubated for four weeks at 37°C and are then read.The presence of 100 or more colonies on 0.2 or 1.0

Htg/ml, or both, of isoniazid is taken as an indicationthat the strain is resistant to isoniazid.

Circumstances for performance of the simple test forisoniazid resistance or of a more accurate test

We recommend that, wherever possible, sensitivitytests be carried out by one of the three " indirect "procedures considered as accurate. The simple testfor isoniazid resistance may be done before the startof antituberculosis chemotherapy as a guidance inthe choice of the regimen either for patients whoclaim not to have received previous chemotherapy orfor those whose treatment is being changed followingthe failure of a previous regimen. If resistance isdemonstrated either by one of the accurate methodsor by the simple test on a strain obtained frompatients who claim not to have received previouschemotherapy, the result should not be taken toindicate that treatment with isoniazid must neces-

British Standard Wire Gauge.

sarily be withheld. However, such a result indicatesthat it is advisable to give additional chemotherapypreferably with at least two drugs other thanisoniazid.

In any survey of the prevalence of resistant tuberclebacilli in a country or territory, the sensitivity testsshould be carried out, whenever possible, in labora-tories equipped and staffed for the purpose of oneof the recommended accurate procedures. However,preliminary information, which must be treated withreserve, can be obtained by the performance of thesimple test for isoniazid resistance.

Future areas for research

With regard to particularly important subjectsfor further research, we make the following recom-mendations:

1. Attempts should be made to improve thestandardization of sensitivity tests and to test theirapplicability in different laboratories. Of importanceis the question of the standardization of the mediaemployed, including the investigation of lyophilizedor dried medium. It is recommended that a com-parison be made between different carefully selectedmethods in each of several laboratories. For thispurpose a single set of strains should be tested ineach laboratory. The methods themselves must bestrictly adhered to in each laboratory.

2. Further research is necessary on rapid methodsof sensitivity testing, since the direct tests mentionedin the present paper require a period of four weeksbefore the result is available.

3. The influence of primary resistance on thetherapeutic progress of patients under chemotherapyis of great interest in assessing the clinical significanceof laboratory tests. In such studies both the degreeof resistance and the proportion of organisms thatare resistant in the bacterial population should bedetermined.

4. Studies should be conducted in different terri-tories on the prevalence of infections due to drug-resistant organisms in patients who claim not tohave received previous chemotherapy. If possible,attempts should be made to subdivide such patientsinto those who failed to disclose previous chemo-therapy at the time when the specimen was obtainedand those who have true primary resistance.

5. Further information on the spread of isoniazid-resistant organisms within the community is urgentlyrequired.

570 G. CANEYTI AND OTHERS

II. THE ABSOLUTE-CONCENTRATION METHOD 1

BASIC OBSERVATIONS

The absolute-concentration technique of deter-mining mycobacterial drug sensitivity has been in usefor some years throughout the world. The authorpresents here a description of the method, as modi-fied by her in respect of some important points.

Preparation and checking of uniform drugconcentrations

The method requires the preparation of uniformdrug concentrations in the culture medium and theircareful checking in each new batch of media. Thischecking should not be confined only to the con-centrations used for testing the strains underinvestigation. In addition, each drug must be testedfor the minimum concentrations in which thereference strain H37Rv or other susceptible strainsisolated from untreated patients are able to multiply.This point is particularly important since the inter-national control strain, H37Rv, is a little moreresistant to isoniazid, and in particular to PAS,than the so-called " wild " strains.

Choice of appropriate size of inoculum

The method also requires particular care inthe choice of the appropriate size of inoculum.Since the composition of a culture of Myco. tuber-culosis is not uniform with regard to the proportionof resistant and sensitive bacilli but correspondsstatistically to a normal distribution (bell-shaped)curve, there will be for each strain some concentra-tions to which all the micro-organisms will beresistant, and others to which only a smaller or largerproportion of them will be resistant. The result, ofcourse, will depend to a large extent on the size ofthe inoculum.

Resistance on the part of the micro-organisms isclinically significant when at least 1% of the totalbacterial population develops at the so-calledcritical concentrations, that is, the weakest concen-trations at which susceptible bacilli are unable to growin the presence of the drug. Any smaller proportion ofresistant micro-organisms has no clinical significance.The inoculum must therefore be selected so that

it is certain to show 1 % of resistant micro-organisms;

1 Prepared by Professor Gertrud Meissner.

to obtain, for example, growth of about 50-100bacilli, the inoculum must contain at least 5000-10 000 bacilli. If the inoculation is performed witha loop (85-100 loops = 1 ml), 1 ml of the bacterialsuspension should therefore contain 500 000-1 000 000 bacilli. If a 0.1-ml capillary pipette isused for the inoculation, the number of micro-organisms in the suspension should not be morethan 50 000-100 000 per ml.

Since it is not possible to determine the viablecounts for each culture to be tested in the course ofroutine drug-sensitivity determinations, the size ofthe inoculum must be chosen with the greatest care.

TECHNIQUE OF RESISTANCE DETERMINATION

Culture medium

L6wenstein-Jensen medium with 0.75% glycerolis used; 5 ml of medium are added to a rimless test-tube or screw-capped test-tube (80 mm long, 27 mmin diameter) and the medium is then coagulated in aslanting position in flowing steam at 82-85°C for40-60 minutes, the time of coagulation being countedfrom the moment when the above temperature isreached. If an autoclave is used, it is possible firstto create a vacuum and then to introduce hot steam;in this case, the time for coagulation is reduced toabout 20 minutes.During coagulation, the rimless test-tubes should

be plugged with cellulose; for storing in the refri-gerator at +4°C, they must be hermetically closedwith rubber bungs to prevent desiccation. If screw-capped tubes are used, the cap should be onlyhalf screwed down during coagulation and screweddown completely only when the tubes are taken out.

After inoculation, the rubber bungs in the test-tubes should be replaced with plugs that will allowentry of a little air. For this purpose, rubber bungswith a spiral perforation or with a strand of silkthreaded through the middle may be employed.

Preparation of the drug dilutions

For isoniazid, streptomycin (i.e., dihydrostrepto-mycin base), and PAS (i.e., pure p-aminosalicylicacid), 1% stock solutions are prepared in distilledwater and the necessary amounts of drug are cal-culated as follows: I g of isoniazid = 1 g; 1 g of

LABORATORY METHODS FOR TESTING DRUG SENSITIVITY OF MYCOBACTERIA 571

dihydrostreptomycin = 1.17 g of dihydrostrepto-mycin sulfate; 1 g of PAS = 1.38 g of sodium PAS.From these stock solutions are prepared the dilutionsto be added to the culture medium in a proportionof 1 : 10 before coagulation. The dilution usedshould therefore be 10 times more concentrated thanthe desired final dilution in the culture medium.For thioacetazone 1 and ethionamide 2 (Ig = 1 g),

a 1% solution in ethylene glycol is prepared. Fromthis stock solution, the dilutions are prepared indistilled water, the pipettes and the distilled waterbeing previously warmed to prevent precipitationof the drug. Immediately after the dilutions havebeen prepared, they should be added to the eggmedium (9 ml of culture medium plus 1 ml of dilu-tion), which is then mixed well and transferred asquickly as possible to the culture tubes. Bothdrugs remain in solution in the egg medium.

Stock solutions of streptomycin, thioacetazone,and ethionamide can be stored in the refrigerator fora limited time in flasks fitted with rubber stoppers.Stock solutions of PAS should be prepared freshlyeach time, and the same procedure is recommendedalso for isoniazid. Final dilutions of all thesedrugs should be prepared freshly for each batch ofmedium.The drugs are added to the culture medium before

coagulation.The drug solutions need not be sterilized by

filtration before they are added to the culturemedium provided that they have been prepared understerile conditions and provided that the drug-containing medium is heated during coagulation.For the determination of drug sensitivity by a

simple standard method, the following drug con-centrations in the medium are recommended:

Drug AdditionalDrug concentrations ~concentrations

gogtmlo for reference strains

Isoniazid .. 0.2 1.0 0.05 0.01Streptomycin

(dihydrostrepto-mycin sulfate) 5.0 10.0 2.0 1.0

PAS . . . . . . . 0.5 2.0 0.2 0.1Thioacetazone . . . 1.0 5.0 0.5 0.1Ethionamide. . 20 50 10 5.0

For each strain tested, two control tubes withoutany drug should be used.

I 4'-formylacetanilide thiosemicarbazone.2 2-(ethyl)thioisonicotinamide.

Preparation of the inoculum

In the case of moist colonies (for example, thosegrown on Gottsacker egg-yolk medium), the bacillimay be ground with the loop directly against thewall of a test-tube containing about 1 ml of Dubosmedium without albumin. If the colonies are drier,they should be ground in a Griffith mortar, alsocontaining Dubos medium without albumin. Theadjustment is made by comparing the turbidity ofthe suspension with that of a standard bariumsulfate solution equivalent to 1 mg/ml of wetbacterial mass. The bacterial suspension obtainedin this way is diluted 1: 50, that is, two drops(0.1 ml) are added to 4.9 ml of Dubos medium.This gives a suspension containing 0.02 mg ofbacteria per ml, i.e., about 200 000-1 000 000organisms per ml. A loopful (diameter: 3 mm) ofthe suspension (85-100 loops = 1 ml) containing2 000-10 000 organisms is used as the inoculum.The inoculum should never be smaller than this,

and may be only slightly larger.

Incubation

The cultures should be incubated for four weeksat 37°C. If the cultures are still not readable,incubation should be prolonged for one to two weeks.

Reading of the cultures

+ + + + = confluent growth, as in the controls.++++ _ discrete colonies according toamount of++J growth.+ = 50-100 isolated colonies.(+) = 20-49 isolated colonies.

Less than 20 isolated colonies is consideredeffective inhibition. The result is considered to benegative if the controls produce profuse growth.A distinction should be made between total

resistance (growth as in the controls) and partialresistance (growth markedly less than in the controls).Finally, note should be taken of any case in whichthere is only a very small number of resistantorganisms.

Evaluation of results

For each drug it is necessary to determine thelowest concentration at which the bacilli may nolonger be considered susceptible, but are to beregarded as resistant (" critical" concentration).

2

G. CANETTI AND OTHERS

This concentration should be determined in eachlaboratory according to the existing experimentalconditions, on a series of so-called " wild " strainscultivated side by side with control strains. Underthe above-mentioned test conditions, the " critical "concentrations are considered to be: 0.2 ,ug/ml forisoniazid; 5 ,ug/ml for streptomycin; 0.5 ,tg/mlfor PAS; 1 ,ig/ml for thioacetazone; 20 ,ug/ml forethionamide. If at these concentrations growth is

observed to the extent of more than 20 colonies,the strain is to be considered resistant.

Communication of results

The laboratory report should include the detailsof the tests and the results of the test for each drug.Sample reports from the Borstel TuberculosisResearch Institute on a susceptible strain (A) and a

resistant strain (B) are shown in Table 1.

TABLE 1DRUG-SENSITIVITY TESTS ON MYCOBACTERIA IN PURE CULTURE ON

LOWENSTEIN-JENSEN MEDIUM

A. Susceptible Strain

Drug concentration | Isoniazid 1 Strepto- PAS Thioaceta- 1 Ethion-in culture medium mycin - _ zone amide

50 zg/ml 0 0 0

20 ,. 0

10 ,,0 0 0 0 0

5 0 ++++

2 ,,0

1 ,,0 0 0

0.5 ,,0

0.2 ,,0

0.05 ,,0

Controls: ++++ ++++

Conclusions: Susceptible to isoniazid, streptomycin, PAS, thioacetazone, and ethionamide

B. Resistant Strain

Drug concentration Isoniazid Strepto- PAS Thioaceta- Ethion-in culture medium mycin zone amide

50 mg/ml +++ +++ 0

20 ++++

10 ,, ++++ ++++ 0 0 ++++5 ,++++ ++++

2 ,,0

1 ,++++ ++ ++++0.5 ,,+++

0.2 ,,++++

0.05,, ++++

Controls: ++++ ++++

Conclusions: Almost completely resistant to 50 Ag/ml isoniazid.Almost completely resistant to 50 mg/ml streptomycin.Partially resistant to 1 pg/ml PAS.Completely resistant to I pg/mi thloacetazone.Completely resistant to 20 g/ml ethionamide.

572

LABORATORY METHODS FOR TESTING DRUG SENSITIVITY OF MYCOBACTERIA 573

III. THE RESISTANCE-RATIO METHOD '

MEDIUM

Sensitivity tests are done on L6wenstein-Jensenmedium without potato starch.2 The drugs areadded before inspissation in the concentrations notedbelow. The medium is dispensed in about 3-mlamounts in half-ounce (14-ml) screw-capped bottlesand is inspissated once for 50 minutes at 85°C.

INOCULUM

The sensitivity tests are set up with an inoculumprepared from the growth on primary diagnosticL6wenstein-Jensen medium slopes, that is to say,they are indirect tests. Tests are set up within twoweeks of the slopes becoming positive (usuallywithin three days), and older growths are subculturedif necessary to ensure that the inoculum is composedof young viable organisms.With a 22 SWG 3 nichrome loop, a representative

sweep from the growth is taken on the loop with avolume, judged by eye, of 2 mm3 (approximately2 mg moist weight of bacilli). The growth taken onthe loop is then discharged into 0.4 ml of sterile dis-tilled water contained in quarter-ounce (7-ml) screw-capped bottles together with six 3-mm glass beads.Standardization of the size of the inoculum isimportant and depends on estimating the amountof growth on the loop as 2 mm3. It is advisablethat the worker should initially weigh a number ofsuch loopfuls of growth to ensure that there isactually 2 mg of growth on his loop. A suspensionis prepared by shaking the quarter-ounce bottle forone minute on a mechanical shaker, and then, witha 3-mm external diameter 27 SWG nichrome loop, aloopful of the suspension is spread on the surfaceof each slope of the sensitivity test.A control drug-free slope is set up for each strain

tested. The standard sensitive strain, H37Rv, istested in each set of tests and again within each setif the batch of medium is changed.

INCUBATION AND READING OF TESTS

The slopes are incubated at 37°C. A reading maybe made at two weeks to give a preliminary indica-tion as to the presence of resistant strains, but the

Prepared by Dr D. A. Mitchison.Jensen, K. A. (1955) Bull. int. Un. Tuberc., 25, 89.

3British Standard Wire Gauge.

final and definitive reading is at four weeks, and areport that a strain is sensitive should not be givenearlier. For all tests, " growth " is defined as thepresence of 20 or more colonies. The resistance ratio(RR) is the minimal concentration inhibiting growth(as defined above) of the test strain divided by theminimal concentration inhibiting growth (as definedabove) of the standard sensitive strain, H37Rv, inthe same set of tests.

SHORT TEST

After the test has been in use for a period, therange of drug concentrations may be shortened withlittle loss of information. The concentrations thatcould be omitted in our tests are indicated with anasterisk below. However, it is possible that slightlydifferent concentrations might be needed in otherlaboratories. The range required for the teststrain is determined by the variation in the minimalinhibitory concentration (MIC) ofH37Rv, and by theneed to determine a resistance ratio of 2 or less forsensitive strains and a resistance ratio of 8 or morefor resistant strains. Thus, if H37Rv is inhibited byeither 8 or 4 ,ug/ml streptomycin in a series ofbatches of tests, then it would be necessary to have8 ,tg/ml as the lowest streptomycin concentration inthe test strain range (test strain MIC = 8 ,ug/ml;H37Rv MIC = 4 ug/ml; RR = 2 or less) and32 ,ug/ml as the highest concentration (test straingrows on 32 ,tg/ml; H37Rv MIC = 8 ,ug/ml; RR =8 or more).

STORAGE

All stock solutions are normally prepared atmonthly intervals and stored at 4°C. Drug-containing media are also stored at 4°C, and canbe used for at least two months after preparation.

PROCEDURE WITH DIFFERENT DRUGS

IsoniazidStock solutions are prepared in distilled water and

sterilized with a membrane, candle or sintered-glass filter, or by very brief autoclaving (5 minutesat 10 pounds per square inch (0.7 kg/cm2)). Seitzfiltration should not be practised as it removes someof the isoniazid.

574 G. CANETTI AND OTHERS

Concentrations in test medium

Test strain: 0.2, 1, 5* and 50* lAg/ml isoniazidH37Rv: 0.025,* 0.05, 0.1, 0.2 and 1 * ,ug/ml isoniazid* Not necessary in short test. However, the inclusion of 50 jAg/ml

is helpful in the identification of atypical mycobacteria, since growthof tubercle bacilli on this concentration is usually catalase-negative,whereas that of atypical mycobacteria is often catalase-positive.

Definition of resistance

Sensitive: no growth (less than 20 colonies) on 0.2<g/ml isoniazid

Resistant: growth on 1 Hg/ml isoniazid

Doubtful: growth on 0.2 but not on I /g/ml isoniazid

(A doubtful test is repeated from the control slope. Ifthe same reading is obtained, or a more resistant one,the strain is finally classified as resistant. If there is nogrowth in the repeat test on 0.2 ,g/ml, the strain isfinally classified as sensitive.)

Streptomycin

Stock aqueous solutions are prepared with asepticprecautions from sterile ampoules of streptomycinsulfate.

Concentrations (before inspissation) in test medium

Test strain: 8, 16, 32, 64* and 1024* ug/ml streptomycinH37Rv: 2, 4, 8 and 16* itg/ml streptomycin* Not necessary in short test.

Definition of resistance

Sensitive: a resistance ratio of 2 or lessResistant: a resistance ratio of 8 or moreDoubtful: a resistance ratio of 4(A doubtful test is repeated from the control slope. If aresistance ratio of 4 or more is obtained, the strain isfinally classified as resistant. If the ratio in the repeattest is 2 or less, the strain is finally classified as sensitive.)

PASStock solutions are prepared from sterile ampoules

of sodium PAS dihydrate.Concentrations in test medium

Test strain: 1,* 2, 4, 8 and 16* jug/ml sodium PASdihydrate

H37Rv: 0.25,* 0.5, 1 and 2 ,tg/ml sodium PASdihydrate

* Not necessary in short test.

Definition of resistance. As for streptomycin.

IV. THE PROPORTION METHOD 1

PRINCIPLE OF THE METHOD

Every wild strain of tubercle bacilli containssome mutants resistant to antibacterial drugs. Thedifference between a resistant strain and a susceptiblestrain is that the proportion of resistant bacteriaamong the total number of bacteria making up thestrain is much higher in a resistant strain than in asusceptible one.

If a resistance test is to enable this proportion tobe calculated, it must provide information on thetotal number of viable bacteria and on the numberof resistant bacteria present in the inoculum. Thisinformation is obtained by seeding dilutions of theinoculum in tubes containing medium without thedrug and in tubes containing medium with the drug.If the dilutions are well chosen, colonies can beobtained in both sets of tubes in numbers that can becounted, and it is easy to deduce from them theproportion of resistant bacteria in the total viablebacterial population.

1 Prepared by Dr G. Canetti and Dr J. Grosset.

THE STANDARD TEST

Culture medium; concentrations of drugs employed;incorporation of drugs into the mediumLowenstein-Jensen medium is used for all the

resistance tests. The tubes used are 17 mm in dia-meter and contain 7 ml of medium. The drugs,dissolved in distilled water, are incorporated in themedium before coagulation. In the standard test,media containing the following drug concentrationsare used:

Isoniazid: 0.2 ,tg/ml and 1 ,ug/mlStreptomycin: 4 ,tg/ml and 10 jug/mlPAS: 0.5 ,g/ml and 1 ,tg/mlThe control medium without the drug is prepared

at the same time as the drug-containing media.In the case of media containing streptomycin,

the drug to be incorporated is dihydrostreptomycinsulfate, not streptomycin sulfate. The susceptibilityor resistance of strains to these drugs is the same, butthe results of the tests are much more consistent whendihydrostreptomycin sulfate is used, because of the

LABORATORY METHODS FOR TESTING DRUG SENSITIVITY OF MYCOBACTERIA

much greater variations in the degree of inactivationof streptomycin sulfate in egg media.

In the case of media containing PAS, the concen-trations given here are for p-aminosalicylic acid. If asodium salt of PAS is used to prepare the solution,1.38 g of the salt are employed for 1 g of p-amino-salicylic acid.

After the drug has been added to the medium andafter the medium has been distributed in the tubes,the medium is coagulated at 85°C for 50 minutes.The tubes are left at room temperature for 24 hourswith cotton-wool plugs and are then covered withrubber caps and put in the refrigerator.

Technique of the indirect test

A spatula measuring 12 mm by 6 mm is used topick off portions containing the greatest possiblenumber of colonies from the primary culture. Theinoculum thus obtained is placed in a sphericalflask, 5 cm in diameter, containing 0.5 ml of distilledwater and 30 glass beads 5 mm in diameter.The flask is shaken by hand for 20-30 seconds;

5 ml of water are added. The bacterial suspensionis then removed and placed in a tube of 17-mmdiameter. The opacity of the suspension is adjustedby the addition of sterile distilled water to correspondto that of a standard suspension containing 1 mg/mlof tubercle bacilli.

Tenfold serial dilutions (10-1 mg/ml, 10-2 mg/ml,etc., down to 10-5 mg/ml) are made from this stocksuspension. They are made in distilled water, theliquid being repeatedly aspirated in the pipette inorder to ensure a homogeneous mixture and thepipette being changed for each dilution. Thedilutions to be inoculated on the media with andwithout the drug are 10-3 mg/ml and 10-5 mg/ml.The inoculum for each tube is 0.2 ml. Two tubes ofmedium without the drug and one tube of mediumwith the drug are seeded with each dilution. Sixteentubes in all are used in a standard test for the threemajor antituberculosis drugs.

After inoculation, the tubes are plugged withcotton-wool but the caps are not replaced. Thetubes are put in a stand at a very slight angle fromthe horizontal and placed in the incubator at 37°C.It is important that the liquid should cover thewhole surface of the medium without, however,touching the cotton-wool plug. When the liquidpart of the inoculum has evaporated (after 24-48hours), the tubes are covered with rubber caps andleft in the incubator at 37°C until the results are read.

The time needed by an experienced technician tocarry out an indirect standard test for the threemajor antituberculosis drugs is 10-15 minutes.

Technique of the direct test

Any specimen in which microscopic examinationdemonstrates a sufficient number of bacilli can beused for a direct resistance test. The techniquedescribed below is the one applied to sputum.Two millilitres of sputum are placed in a sterile

mortar. An equal quantity of4% NaOH and 3 dropsof tincture of litmus are added. The mixture istriturated with the pestle for one or two minutesand then wrapped in sterile paper and incubatedat 37°C for 50 minutes.A smear of the sputum specimen is prepared

beforehand. During incubation of the sputum, thesmear is stained by the Ziehl-Neelsen method andexamined under the microscope.

1. If the smear is found to contain less than onebacillus per 10 immersion-objective fields, or nobacilli at all, the specimen is subjected to the indirecttest described above.

2. If the smear proves to contain at least onebacillus per 10 fields, the specimen is subjected to adirect test, as described below.The sputum is taken from the incubator after

50 minutes, and not centrifuged. It is then neutra-lized with a few drops of 15% sulfuric acid. Theproduct thus obtained constitutes Dilution 1.Further dilutions are prepared in tenfold serial steps.The dilutions to be used for inoculation depend onthe number of bacilli seen under microscopicexamination.

1-10 bacilli per 10 immersion-objective fields.Dilution 1 and Dilution 10-2 are inoculated on thedrug-containing slopes and on the control slopes(without drug).

1-10 bacilli per immersion-objective field. Thecontrol tubes and drug-containing tubes are inocu-lated with Dilutions 1 and 10-2. The control mediaonly are inoculated with Dilution 10-3.

10 bacilli per immersion-objective field. The drug-containing tubes and control tubes are inoculatedwith Dilutions 10-1 and 10-8.

The inoculum for each tube is 0.2 ml. The numberof tubes inoculated per dilution and the procedureafter inoculation are the same as in the indirect test.

575

0. CANETI AND OTHERS

Reading the results; criteria of resistanceThe results are read for the first time on the

28th day. If the results of the test for sensitivity to agiven drug are such that, according to the criteriaindicated below, the answer is " strain resistant ", nofurther reading of the test is required. If the strainis shown to be sensitive, a second reading, made onthe 40th day, will provide the definitive answer.

In the tubes inoculated with the lowest dilutionthat is still positive the number of colonies is care-fully counted. It is important that even very smallcolonies should be counted. The number of coloniesobtained in the control tubes (average of the twotubes) indicates the number of viable bacillary unitscontained in the inoculum for the correspondingdilution. The number of colonies obtained in thetube containing the drug indicates the number ofresistant bacillary units contained in the sameinoculum.The ratio between the second figure and the first

shows whether the strain is susceptible or resistant.Results are read in the same way in the direct andindirect tests.The criteria of resistance are as follows:Isoniazid. Any strain containing at least 1 % of

bacilli resistant to 0.2 ,ug/ml of isoniazid or a higherconcentration is classified as isoniazid-resistant.

Streptomycin. Any strain containing at least 1%of bacilli resistant to 4 ,ug/ml of dihydrostrepto-mycin or a higher concentration is classified asstreptomycin-resistant.PAS. Any strain containing at least 1 % of

bacteria resistant to 0.5 ,ug/ml of PAS or a higherconcentration is classified as PAS-resistant.

It may happen in a direct test that less than 100colonies are obtained with the lowest dilution(generally Dilution 1) in the tubes not containingthe drug. In such cases, if the tubes containing theweakest concentration of the drug do not yield anycolony, an indirect test should be made. The fre-quency with which this happens varies according tothe material studied: it is in the region of 10-15 % oftests. It is very rare in the indirect test.

OTHER TYPES OF TEST BY THE PROPORTION METHOD

The standard test described above answers theneeds of routine practice. Two other types of testfor which a direct or indirect technique can be usedcan be performed by the proportion method. One ismore complex-the "population study test"-while

the other is simpler and is known as the " economicaltest ".

Population study test

For research purposes it may be useful to knowwhat proportion of mutants with very different levelsof resistance exists in a strain of Myco. tuberculosis.In that case a wider range of concentrations of thedrug in the medium must be used. Media containingthe following drug concentrations are then recom-mended:

Isoniazid.DihydrostreptomycinPAS.

pg ofdrug per mlofmedium

0.1 0.2 1 52 4 10 1000.25 0.5 1 10

Furthermore, if an indirect test is being performed,a bacterial dilution of 10-1 mg/ml should be seededas well as the dilutions of 10-3 mg/ml and 10-5 mg/ml.Forty-two tubes are needed for a test of this kind.

Economical testIt is possible to perform a valid resistance test

using a smaller number of tubes than that indicatedfor the standard test. With this in mind, only onecontrol tube is inoculated with each of the twobacterial dilutions of 10-3 mg/ml and 10-5 mg/ml.Furthermore, the tubes containing the higher con-centrations of the drugs are eliminated in eachdilution (the tubes kept are 0.2 ,ug/ml of isoniazid,4 ,ug/ml of dihydrostreptomycin, and 0.5 ,ug/ml ofPAS). The number of tubes needed for the econo-mical test is eight. It should be emphasized that it isimpossible in any case to dispense with the inocula-tion of one or other of the two bacillary dilutionsof 10-3 and 10-5 mg/ml and that the same care shouldbe given to the preparation of these dilutions as in thestandard test.

TEST OF RESISTANCE TO SECONDARYANTIBACTERIAL AGENTS

The susceptibility of strains of Myco. tuberculosisto what are known as the " secondary " antibac-terials (ethionamide, cycloserine, viomycin, kana-mycin and thioacetazone is measured in thesame way as susceptibility to the major antituber-culosis drugs. Information on the concentrationsof the drug to be incorporated in the medium andthe minimum proportions of resistant bacilli thatmust be found on the critical concentration of eachdrug if the strain tested is to be considered resistantcan be obtained on request from the authors.

576

LABORATORY METHODS FOR TESTING DRUG SENSITiVITY OF MYCOBACTERIA

TABLE 2NUMBER OF MUTANTS RESISTANT TO VARIOUS CONCENTRATIONS OF

THE THREE MAJOR ANTITUBERCULOSIS DRUGS FOUND AMONG 10' BACILLI OFWILD STRAINS OF MYCO. TUBERCULOSISa

A. Isoniazid

Number of I. gm . s/l Iu/l 5;gmresistant mutants 0.1 iigml 0.2 gmI | 1.gImI 55sgmI

Minimum 2 0.5 0 0

Maximum 180 41 12 7

Average 41 5 3.3 1.5

B. Dihydrostreptomycin

Number of 1 g/l 4 u/l 10 A1 1 0,gmresistant mutants 2MgIml | gIm | 10 sgm | 200 gmlb

Minimum 100 0.7 0 0

Maximum 100 000 400 12 1

Average >5 000 c 41 2 0.1

C. PAS

Number of 0.25 g/ml 0.5 gg/ml I g/ml 10 pg/mlresistant mutants

Minimum 0.1 0 0

Maximum 11 000 100 32 8

Average 800 c 25 4.6 0.7

a Drugs Incorporated in L5wenstein-Jensen medium before inspissation; results read on the40th day.

b The concentration now advised is 100 JAg/mi.c Median value.

CONTROL TESTS TO BE CARRIED OUT IN A LABORATORYPERFORMING SUSCEPTIBILITY TESTS BY THE

PROPORTION METHOD

These control tests are necessary in two circum-stances: first, when a laboratory begins to use theproportion method and, secondly, every time a newbatch of drug-containing media is made in thelaboratory or is received from outside.

Preliminary test required when the proportion methodis first usedIn these circumstances, it is strongly recommended

that the method be tried out on a series of five wildstrains. These strains should preferably be requestedfrom a reference laboratory; otherwise strains couldbe used that have been isolated in the laboratoryitself from patients who are known for certain neverto have been treated with antituberculosis drugs andnever to have been in contact with treated tuber-culosis patients. These five strains, cultivated on

Lowenstein-Jensen medium, are subjected to a specialindirect test known as the " control test ". Theinoculum is obtained and dilutions (down to 10-6 mg/ml) are prepared in strict conformity with the methodalready described above. The concentrations ofdrugs in the media to be used for the control testare the same as those indicated for the standard test,namely:

,Lg ofdrug per mlof medium

Isoniazid.DihydrostreptomycinPAS.

0.2 14 100.5 1

The following bacillary dilutions are seeded inone tube not containing a drug and in one tube foreach drug concentration: 1 mg/ml; 10-1 mg/ml;10-2 mg/ml.The following bacillary dilutions are seeded only

in two tubes not containing a drug: 10-3 mg/ml;10-5 mg/ml; 108-mg/ml.

577

G. CANETfI AND OTHERS

The total number of tubes needed for this controltest is 27 per strain. The final results are those readon the 40th day and are expressed as the number'ofresistant bacilli present among 106 viable bacilli ofthe strain studied.The figures found for each strain should be com-

pared with those given in Table 2. In at leastfour of the five strains tested, they should fall, foreach drug concentration, between, or very close to,the minimum and maximum values given in thetable for the corresponding drug concentration.

Control test required when a new batch of media isprepared or receivedIn these circumstances, a control test identical

with that described above should be performed on

Depuis l'introduction des m6dicaments antituber-culeux, de grands progres ont ete faits dans la lutte contrela tuberculose et d'autres maladies infectieuses. Cepen-dant, l'activit6 de ces substances est limit6e par l'appari-tion de souches bacteriennes capables de se d6velopperen presence de fortes concentrations de ces medicaments.On connait maintenant des souches de bacilles tuber-culeux r6sistantes A chacun des principaux medicamentsantituberculeux. I1 arrive meme que certaines souchessoient naturellement resistantes A une substance chimique- avant d'avoir ete mises en contact prolonge avec elle.C'est ainsi que le bacille tuberculeux bovin peut etrenaturellement r6sistant a l'acide p-aminosalicylique. Onevalue a 3-15% le nombre de souches provenant demalades non traites qui se revelent resistantes a un ouplusieurs des principaux medicaments antituberculeux(isoniazide, streptomycine, PAS).Le Comite OMS d'experts des Antibiotiques a estime

que, pour sauvegarder l'efficacite - a l'echelle del'individu et de la collectivite -des medicaments anti-tuberculeux, il etait essentiel de determiner la sensibilitedes souches de bacilles tuberculeux A ces substances.Car, un des problemes majeurs des campagnes contre latuberculose fond6es sur la chimiotherapie de masse estla dissemination de bacilles resistants aux medicaments.

Les m6thodes de determination et d'appr6ciation de lasensibilite etant diverses et variees, et leur uniformisationrepr6sentant une premiiere etape, fondamentale, vers laconnaissance des ph6nom6nes de resistance, un groupede sp6cialistes en bacteriologie de la tuberculose a etreuni par l'OMS. Cet article donne un aperqu de leursdiscussions et un r6sum6 de leurs recommandations.

Les tests de laboratoire relatifs Ala sensibilite du bacilletuberculeux aux medicaments ont trois buts principaux:1) donner une indication pour le premier traitementchimioth6rapique A appliquer au malade; 2) confirmer-

one wild strain. The same strain should always beused. It should be one of the five strains studied inthe preliminary test, preferably one which did notgive extreme values for any of the three drugs.Between tests, the strain should be maintained bymonthly subculture on Lowenstein-Jensen medium.It is quite unnecessary to perform a control test on awild strain for each new series of resistance tests.

Note. If the laboratory intends, in certain cases,to use the " population study test ", the completerange of media used in that test should be used forthe initial as well as for the routine control tests.The bacillary dilutions to be used for inoculationare those indicated above.

;UM1ou infirmer -l'apparition de la r6sistance bacillairelorsqu'un malade n'a pas r6agi favorablement au traite-ment; 3) 6valuer l'existence d'une resistance naturelleou acquise des bacilles tuberculeux dissemines dans lacollectivit6. Les microbiologistes devraient disposer detechniques suires. Malheureusement, plusieurs m6thodessont en usage qui manquent de pr6cision et ne permettentpas de distinguer les micro-organismes sensibles desresistants.En vue de la standardisation des tests de sensibiite,

trois types de methodes ont e proposes:1. methode des concentrations absolues2. methode des rapports de resistance3. methode des proportions.Ces methodes sont decrites en detail dans l'article.Dans leurs recommandations, les participants cons-

tatent qu'il n'est pas possible de recommander, & l'issuede cette premiere r6union, une seule methode standard,et que chacune des trois m6thodes envisag6es repr6senteun compromis. Mais quelques caractWres g6n6raux doi-vent etre retenus pour les tests de sensibilit6.Le milieu doit etre celui de Lowenstein-Jensen, sans

f6cule de pomme de terre (milieu de l'Union inter-nationale contre la Tuberculose). Les solutions dessubstances chimioth6rapiques doivent etre dilu6es dansl'eau distillee. La concentration (surtout s'il s'agit destreptomycine) sera celle reellement ajout6e au milieu.On ne tiendra pas compte de son 6ventuelle inactivationpartielle pendant la coagulation du milieu. Le milieupeut etre conserv6 a +4°C pendant trois mois au maxi-mum. On cherchera a uniformiser les diverses 6tapesdes tests, en particulier en donnant les r6sultats parrapport a des souches . sauvages * de bacille tuberculeuxet en indiquant la teneur de l'inoculum en bacillesviables.

578