APPLIED MICROBIOLOGY, May 1974, p. 917-919Copyright 0 1974 American Society for Microbiology

Vol. 27, No. 5Printed in U.S.A.

Comparison of Two Commercial Formulations of theMacConkey Agar Test for Mycobacteria

GEORGE P. KUBICA AND JACK VITVITSKYTrudeau Institute, Inc., Saranac Lake, New York 12983

Received for publication 21 January 1974

Recent evaluations of the MacConkey agar test for differentiation of rapidlygrowing mycobacteria have revealed that certain strains of Mycobacteriumfortuitum and M. chelonei that were expected to grow on MacConkey agar failedto do so. Investigation of two formulations of MacConkey agar showed that thesetwo species grew better on the medium when the crystal violet dye was omitted.Several possible reasons for this difficulty are discussed. It is recommended thatclinical laboratories engaged in differential identification of mycobacteria utilizecommercial MacConkey agar without crystal violet when testing rapidly growingspecies of this genus.

The differential inhibition of certain myco-bacteria by MacConkey agar (2) has been onemethod of value in the taxonomic separation ofrapidly growing acid-fast bacilli (2, 3, 5, 6, 10).Mycobacterium fortuitum (9) and the twosubspecies of M. chelonei (6), commonly re-garded as the potentially pathogenic fast-grow-ing mycobacteria, usually grow on MacConkeyagar, whereas the normally saprophytic speciesare inhibited by this medium.A recent evaluation of clinical laboratories in

Oregon (Charles E. Gardner, personal commu-nication) revealed that those laboratories thatutilized MacConkey agar without crystal violetgenerally observed growth of the species M.fortuitum, whereas those laboratories employ-ing the same medium with the dye often failedto grow this organism.

Stanford et al. (10) reported some strains ofM. chelonei, notably those observed in Europe,grew poorly if at all on MacConkey agar,whereas strains isolated in the United Statesgrew well on the medium. Observations in ourown laboratory revealed that a number ofstrains that earlier had grown on MacConkeyagar were now unable to do so. Many of thesestrains formerly had been segregated as RapidGrower A (3), a group closely related to M.peregrinum, which is a species now incorpo-rated with M. fortuitum (6). Because a numberof Rapid Grower A isolates were inhibited bythe mixture of methylated pararosanilines re-ferred to as methyl violet (3), and because themore purified hexamethyl pararosaniline crys-tal violet (7) is a component of certain formula-tions of MacConkey agar, we reasoned that

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either the dye or the present inclusion of M.peregrinum in the taxon fortuitum (or a combi-nation of both) might be contributing to thecurrently recognized failures of the once-reliableMacConkey agar test for differential identifica-tion of certain rapidly growing mycobacteria.

In an attempt to quickly reinstate the validityof the MacConkey agar test, before more time-consuming studies would hopefully reveal thetrue reason for its failure, the present study wasundertaken to determine if MacConkey agarwithout crystal violet could be substituted forthe dye-containing medium with a restorationof expected differential growth patterns formycobacteria.

MATERIALS AND METHODSMedia. Since we became aware of this problem,

comparative studies on selected strains of rapidlygrowing mycobacteria have been carried out on fourdifferent lots of MacConkey agar with crystal violet(MCV) obtained from two commercial sources, as wellas three lots of MacConkey agar without crystal violet(MA). Our largest comparative study, presented here,was conducted with media from only one supplier, butcomparable results (with fewer strains) have beenobserved on all preparations of this medium. Mac-Conkey agar (Difco) with and without crystal violet(both of which represent a modification of the originalformula of MacConkey [81), were prepared accordingto directions and poured into plastic petri dishes (15by 100 mm) as described earlier (4).

Cultures. Sixty-six cultures of mycobacteria, ob-tained either from the authors' collection (TrudeauMycobacterial Culture Collection) or from a previousinternational cooperative study (6), were examined.The species distribution was as follows: five strains ofM. chelonei and 10 of M. chelonei subsp. abscessus

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KUBICA AND VITVITSKY

(6); 25 strains of M. fortuitum of which 13 previouslywere identified as M. peregrinum; two strains of M.diernhoferi; 6 of M. flavescens; 3 of M. phlei; 8 of M.smegmatis; and 7 strains of M. vaccae.

Test procedure. For each strain tested, one 3-mmloopful of organisms, obtained from a 7-day culture inenriched Middlebrook 7H-9 broth (Difco), was inocu-lated to both plates of MacConkey agar, using thepetri dish turntable method (3, 4). All plates wereincubated at 37 C in polyethylene bags; some fewstrains, which appeared to grow better at 33 C, wereincubated at both temperatures. Plates were exam-ined from 5 to 14 days, and growth was recorded asbefore (3). Cultures that failed to react as expectedwere retested to confirm the observed results. Platesof Dubos oleic-albumin agar (Difco) inoculated witheach culture served as controls on colony type of testorganisms.

RESULTS AND DISCUSSIONThe comparative growth of all cultures on the

two preparations of MacConkey agar is summa-rized in Table 1.

Forty of the 66 cultures tested (25 strains ofM. fortuitum and 15 strains of M. chelonei)were expected to grow on MacConkey agar. Ofthese, 28 strains (15 of M. fortuitum and 13 ofM. chelonei) grew on both types of MacConkeyagar, while five strains (three M. fortuitum andtwo M. chelonei) grew on neither medium.Seven strains grew only on MA. All three of thestrains of M. fortuitum that failed to grow oneither agar, and five of the seven that grew onlyon MA, had been segregated earlier as M.peregrinum (now part of the taxon M.fortuitum). The former taxon M. peregrinum,closely related to bacilli once referred to asRapid Grower A, had been shown earlier to beinhibited by a related methyl violet dye (3);hence, we were not too surprised by theseresults.

TABLE 1. Comparison of growth of mycobacteria onMacConkey agar with and without crystal violeta

No crystal violet

Growth No growth

Crystal No M. fortuitum (7) M. chelonei (2)violet growth M. smegmatis (2) M. fortuitum (3)

M. diernhoferi (2)M. flavescens (6)M. phlei (3)M. smegmatis (6)M. vaccae (7)

Growth M. chelonei (13)M. fortuitum (15)

'The number of strains which grew, or failed to grow, onthe two kinds of MacConkey agar are indicated parentheti-cally after each species name.

The important observation was that sevenstrains of M. fortuitum grew only on MA, andthese results were significant at the 5% level bythe chi-square test. Thus, for the diagnosticbacteriology laboratory, which is often asked tocomment on the potential clinical significanceof patient isolates, it is recommended that MAbe used in place of MCV until more definitiveevidence reveals the true reason for the failureof MCV in recent trials.Such action is not without problems, how-

ever, for it can be seen that two strains of M.smegmatis also grew, even though poorly, onMA. The diagnostic difficulties posed by therare isolation from man of M. smegmatis maybe obviated by the concomitant performance ofa 3-day arylsulfatase test; almost all strains ofM. fortuitum and M. chelonei are positive inthis test, whereas M. smegmatis rarely, if ever,is able to hydrolyze the substrate (5, 6, 11).

It is difficult to ascertain why some strains ofM. fortuitum and M. chelonei no longer grow onMCV; however, it is interesting to draw frompast experience and to speculate on certaincomponents of the media as likely contributorsto the problem. Examination of the formulas forMCV and MA reveals differences in content orcomposition of the following: peptones, bilesalts, neutral red, agar, and final pH. Althoughthe composition of peptones varies between thetwo formulations, the total quantity (2%) is thesame in both media. The pH of both prepara-tions used was within 0.1 U of the valuerecorded on the label, and because Chapmanand Bernard (1) had shown the rapidly growingmycobacteria to grow quite well over a widerange of pH, it seemed unlikely that pH was afactor. In our experience, most rapidly growingacid-fast bacilli grow well in agar concentra-tions ranging from 1.2 to almost 2.0; because theamount of agar in both MacConkey prepara-tions falls within this range, agar seemed anunlikely factor. Because MA contains a largeramount (0.5%) of bile salts than MCV (0.15%)and because M. smegmatis and M. phlei willnot grow on MA but will grow in 1% deoxycho-late (6), this content of bile salts seemed unim-portant. Earlier we had observed (3) that allstrains of mycobacteria tested grow so similarlyon agar medium containing 0.01% neutral redthat this dye was not considered for differentialidentification of mycobacteria. Because themaximum content of this dye (0.005%) presentin MA was only 50% of the amount tested earlier(3), we have tended to disregard its importanceas an inhibitory agent in MacConkey medium.However, the possibility that subinhibitory lev-els of neutral red, in combination with crystal

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violet, are inhibitory is tenable and should beexplored. Additionally, other combinations ofthe above ingredients taken together couldaccount for the undesirable inhibitory effectobserved in MCV, but we have not yet testedthe multitude of combinations of media thatwould derive with six variables.Our earlier observation (unpublished) that

different lots of the same dyes (which varied inactual dye content) could yield different inhibi-tory growth patterns for mycobacteria was whatprompted us to report the lot number and dyecontent of each product studied (3). For thisreason we have preferred to take the moresimplified view that the current inhibition onMCV of organisms in the M. fortuitum-M.chelonei group is due to a combination of twofactors: (i) inclusion of M. peregrinum in thetaxon fortuitum, and (ii) a possible change incontent or composition of the crystal violet dye.Earlier studies (3) indicated that a group oforganisms closely related to M. peregrinum(i.e., Rapid Grower A) were more easily inhib-ited by MCV and the methyl violet dye thanwas M. fortuitum. The present study included12 strains, labeled M. peregrinum, which hadbeen examined 5 years ago. At that time 10 ofthe 12 grew on MCV, whereas today only 5 of 12grow on MCV, although 10 of 12 grow on MA.Additionally, 30 of the 40 strains of M. for-tuitum-M. chelonei in the present study werealso examined 5 years ago. In 1969, 28 of these30 strains (93%) grew on MCV; today only 63%grow on MCV, although 90% grow on MA.The sudden and unexplained variability of a

single test is rarely of great concern to thetaxonomist who may employ more than 100 testfeatures for species characterization. In con-trast, such test failure could have dramaticconsequences in a diagnostic laboratory, whichnormally performs only a few selected buthighly reliable differential tests (5, 10, 11). Forthis reason, the diagnostic laboratory shouldutilize at least two definitive tests to identifyeach clinically significant Mycobacterium, inthe hope that the failure of one test (as was thecase here with MacConkey agar) could bedetected by another reliable test (in this in-stance the 3-day arylsulfatase activity).

In conclusion, for reasons that still remainnebulous, certain recent batches of MacConkeyagar with crystal violet have been found to yield

spurious results when used for differential iden-tification of mycobacteria. Until such time asmore definitive data may establish reasons forthis failure, it has been shown that MacConkeyagar without crystal violet will give almost thesame mycobacterial growth inhibition patternsas diagnostic laboratories have come to expectof the rapidly growing acid-fast bacilli. Thus,the change to a dye-free medium would serve atleast as a temporary stop-gap measure thatcould renew our faith in this test. For thisreason it is recommended that MacConkey agarwithout crystal violet be used to screen rapidlygrowing mycobacteria, and that appropriatecontrols (known positive and negative reactors)be included with each test run.

ACKNOWLEDGMENTThis study was supported in part by Public Health Service

contract no. AI-80640 (formerly PH 43-68-640) from theNational Institute of Allergy and Infectious Diseases.

LITERATURE CITED1. Chapman, J. S., and J. S. Bernard. 1962. The tolerances

of unclassified mycobacteria. I. Limits of pH tolerance.Amer. Rev. Resp. Dis. 86:582-583.

2. Gordon, R. E., and J. M. Mihm. 1959. A comparison offour species of mycobacteria. J. Gen. Microbiol.21:736-748.

3. Jones, W. D., Jr., and G. P. Kubica. 1963. The differen-tial typing of certain rapidly growing mycobacteriabased on their sensitivity to various dyes. Amer. Rev.Resp. Dis. 88:355-359.

4. Jones, W. D., Jr., and G. P. Kubica. 1964. The use ofMacConkey's agar for the differential typing of Myco-bacterium fortuitum. Amer. J. Med. Technol.30:187-190.

5. Kubica, G. P. 1973. Differential identification of myco-bacteria. VII. Key features for identification of clini-cally significant mycobacteria. Amer. Rev. Resp, Dis.107:9-21.

6. Kubica, G. P., I. Baess, R. E. Gordon, P. A. Jenkins, J. B.G. Kwapinski, C. McDurmont, S. R. Pattyn, H. Saito,V. Silcox, J. L. Stanford, K. Takeya, and M. Tsuka-mura. 1972. A cooperative numerical analysis of rapidlygrowing mycobacteria. J. Gen. Microbiol. 73:55-70.

7. Lillie, R. D. 1969. H. J. Conn's biological stains, 8th ed.The Williams and Wilkins Co., Baltimore.

8. MacConkey, A. 1905. Lactose-fermenting bacteria infaeces. J. Hyg. 5:333-379.

9. Runyon, E. H. 1972. Conservation of the specific epithetfortuitum in the name of the organism known asMycobacterium fortuitum da Costa Cruz. Request foran opinion. Int. J. Syst. Bacteriol. 22:50-51.

10. Stanford, J. L., W. J. Gunthorpe, S. R. Pattyn, and F.Portaels. 1972. Studies on Mycobacterium chelonei. J.Med. Microbiol. 5:177-182.

11. Wayne, L. G., and J. R. Doubek. 1968. Diagnostic key tomycobacteria encountered in clinical laboratories.Appl. Microbiol. 16:925-931.

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