JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1993, p. 2178-2184 Vol. 31, No. 80095-1137/93/082178-07$02.00/0Copyright © 1993, American Society for MicrobiologY

Timely Culture for Mycobacteria Which Utilizes aMicrocolony Method

DAVID F. WELCH,1* ARTHUR P. GURUSWAMY,1 SANDRA J. SIDES,' CHARLES H. SHAW,2AND MARY J. R. GILCHRIST3

Clinical Microbiology Laboratories, University of Oklahoma Health Science Center, Box 26307, OklahomaCity, Oklahoma 731261; Veteran's Affairs Medical Center, Cincinnati, Ohio 452202; and Department

ofPathology, University of Cincinnati, Cincinnati, Ohio 452293

Received 16 February 1993/Accepted 14 May 1993

For the isolation of mycobacteria from clinical specimens, we evaluated a method that used a thinly pouredMiddlebrook 7H11 agar plate (10 by 90 mm) that was examined microscopically. Inoculated plates were sealed,incubated, and examined at regular intervals for the appearance of microcolonies. Plates were examinedmicroscopically, while still sealed, by focusing on the agar surface through the bottom of the plate and the agar.Plates were scanned at low power (x40 total magnification), and colony morphology was confirmed atintermediate power (x 100 to x 180 magnification). This method was compared with a traditional method thatused macroscopic examination of standard mycobacterial media. By using all specimens submitted formycobacterial culture over the duration of the study, the method was evaluated until 270 isolates ofmycobacteria (Mycobacterium tuberculosis, n = 103; M. avium-M. intracelulare, n = 115; miscellaneous, n =52) were detected. While the conventional method required an average of 23 days to the time of first detectionof mycobacteria, the experimental method required an average of only 11 days. When limited to acid-faststain-positive specimens that were culture positive for M. tuberculosis, the average interval to positivity was 7days for the microcolony method compared with 17 days for the conventional method. With the experimentalmethod, the microscopic colonial morphology allowed for the presumptive identification of M. tuberculosiscolonies, which were distinguished by cording, and M. avium-M. intracellulare colonies, which were smoothand entire. Presumptive identification was complete for 83.5% of the M. tuberculosis isolates within 10 days andfor 85% of the M. avium-M. intracelulare isolates within 11 days after inoculation. If the microcolony methodwas combined with a conventional tube medium, the composite would optimize for speed of recovery whileproviding the full sensitivity of the conventional method. In addition to reducing the interval to positivity, themicrocolony method allows for the easy detection of mixed mycobacterial infections and yields a presumptiveidentification that facilitates the selection of a confirmatory gene probe test.

Methods to facilitate the rapid diagnosis of tuberculosishave long been sought (2). During the 1960s there was a greatdeal of activity focused on the use of a clear agar mediumand microscopic evaluation of colonial morphology (11)which was refined by work published in the 1970s (7). Anumber of laboratories elected to adopt this methodology fordetection of these agents in cultures of human clinicalspecimens. The subsequent introduction of a radiometricmethod (BACTEC) for the detection of mycobacteria thatoccurred concomitantly with a decreasing incidence of tu-berculosis in the United States led to a decline in interest inoptimizing traditional cultural methods for the detection ofmycobacteria. However, with the rise in the incidence oftuberculosis in the United States beginning in the mid-1980s,the subsequent recognition of multiply drug-resistant strainsof Mycobacterium tuberculosis, and the surge in AIDS-associated M. avium-M. intracellulare (MAI) infections hascome the renewed recognition of the need to optimize therapid detection of mycobacteria. Pending the widespreadintroduction of satisfactory genetic amplification methods,which are now under active investigation, the laboratorycommunity must expeditiously adopt alternative rapid meth-ods.While the radiometric method has become the mainstay

* Corresponding author. Electronic mail address: f710@rex.re.uokhsc.edu.

among rapid culture methods for mycobacteria, it has anumber of limitations that inhibit its universal application.First, since it involves automated, serial sampling of bottles,fastidious attention to preventive maintenance is necessary inorder to prevent the transfer of microorganisms from positiveto negative bottles and subsequent pseudomycobacterial in-fections. Second, since it entails the utilization of radioiso-topically labelled substrates, institutions in some jurisdictionsmay experience difficulty conforming to local requirementsfor monitoring and disposal of isotopes, which are morepronounced in the United States with the 1993 radioactivewaste disposal initiatives (on the basis of the deadline forstates to conform to the 1985 amendments to the 1980Low-Level Radioactive Waste Disposal Act). Third, themethod may be too technologically demanding for laborato-ries in many settings, particularly in developing countries.Finally, the instrument may not be readily available orcost-effective for implementation in some settings. Thus, themicrobiology community could greatly benefit by the optimi-zation of an alternative method that would bridge the currenttechnology gap to future rapid technologies. The microcolonymethods devised in the 1960s and refined as detailed hereinrender this technology suitable for filling that void.

MATERIALS AND METHODS

Over a 30-month period, all nonblood specimens submit-ted for mycobacterial culture in the two institutions were

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TABLE 1. Comparison of recovery yield and rate by the microcolony method and with conventional media for isolation ofmycobacterial species

Species isolated (no.) % Acid-fast No. (%) of isolates detected by: Avg interval (days [range]) to first detection by:stain positive Microcolony method Conventional method Microcolony method Conventional method

M. tuberculosis (103) 62 86 (84) 102 (99) 10 (3-28) 21 (9-46)M. avium complex (115) 50 98 (85) 102 (89) 11 (3-42) 25 (6-50)M. kansasii (23) 61 22 (96) 22 (96) 7 (2-15) 19 (6-38)M. gordonae (14) 0 10 (71) 10 (71) 23 (9-31) 30 (17-38)M. fortuitum complex (9) 33 6 (33) 9 (100) 3 (2-11) 11 (3-20)M. scrofidaceum (3) 0 1 (33) 3 (100) 9 38 (33-45)Other (3)a 0 2 (67) 3 (100) 6 (4-11) 31 (13-56)

Overall (270) 51 225 (83) 251 (93) 11 (2-42) 23 (3-56)

a One each of M. xenopi, M. terrae, and Nocardia sp.

cultured in parallel by both a conventional method, applyingmacroscopic examination, and the microcolony method,relying on the use of plated media and microcolony detec-tion. Specimens from the respiratory tract and tissue consti-tuted 83 and 16% of the samples, respectively. Specimensexpected to contain other microbial agents were subjected toa routine protocol for digestion and decontamination withN-acetylcysteine and sodium hydroxide. When of sufficientvolume, fluid specimens were concentrated by centrifuga-tion at 3,000 x g for 30 min. All manipulations of specimens,when vials were opened, were conducted in a certifiedbiosafety cabinet to contain the aerosols that were poten-tially generated by adding reagents. When tubes were mixedby vortexing, the lids were sealed and the vortexing opera-tions were performed in the biological safety cabinet. Cen-trifugation of specimens was conducted in sealed safetycarriers, and these carriers were opened in the biosafetycabinet. By using the resuspended pellet from these proce-dures, an inoculum of 0.1 ml of each was inoculated onto oneof each of the following media: Lowenstein-Jensen (BBL,Cockeysville, Md.) slanted in tubes, selective Middlebrook7H11 (BBL) slanted in tubes, and a thinly poured (18 ml)plate (10 by 90 mm) of Middlebrook 7H11 (Remel, Lenexa,Kans.) that was subsequently fitted with a Shrink-Seal(Scientific Device Laboratories, Glenview, Ill.). After inoc-ulation, all media were incubated at 35°C in an atmospherecontaining 5 to 8% carbon dioxide. Plated media wereincubated for a total duration of 4 weeks, and tubed mediawere incubated for a duration of 6 to 8 weeks.

Microscopic evaluation of the sealed plates was conductedby focusing through the bottom of the plate and the agar; thecorrect plane of focus was easily identified by focusing on

the streak lines still evident in the agar surface. The mediawere screened twice weekly. Tubed media were evaluatedmacroscopically and plated media were examined micro-scopically by scanning the agar surface encompassing thefirst two streaked quadrants at x40 magnification. Whendetected, colonies were evaluated at x 100 to x 160 totalmagnification to determine their colonial morphologies.While an American Optical compound microscope was usedeffectively for this activity, a Zeiss or Olympus invertedmicroscope was more efficient. Microcolonies were charac-terized according to the descriptive terms relating to theirconsistency (cording, granular, or smooth) and their margin(entire, irregular, diffuse, compact, or spider). Throughoutall of the microscopic screenings, the seals remained intact,with the lids of the plates securely in place. When suspectcolonies were detected microscopically, the plates were

taken to a biosafety cabinet, where the seals were removed.Colonies were picked and stained for acid fastness, and ifthey were acid fast, they were subcultured for confirmatoryidentification by conventional methods. All plates wereresealed before being removed from the biological safetycabinet.

RESULTS

The recovery of mycobacterial species by the experimen-tal single-plate microcolony method compared with therecovery of mycobacteria by the conventional two-tubemacroscopic method is depicted in Table 1. The comparativeoverall detection rates were 83% (microcolony method) and93% (conventional method), respectively. Approximately85% of the M. tuberculosis and MAI isolates were detectedby the single-plate microcolony method. With the exceptionof M. kansasii, other mycobacterial species were detected

TABLE 2. Mycobacterial isolation by microcolony andconventional methods stratified by smear results

Acid-fastInterval to positivity (days) by:

stain result Species (no.) Microcolony Conventionalmethod method

Negative MTBa (15) Negativeb 33bMTB (24) 16 24

Rare MTB (3) Negativeb 23bMTB (37) 7 16

Few MTB (5) 6 16Moderate MTB (6) 7 19Many MTB (8) 9 19

Negative MAI (9) Negative 35bNegative MAI (41) 15 24Rare MAI (5) Negative 29bRare MAI (31) 8 18Few MAI (7) 7 19Moderate MAI (1) 7 16Many MAI (12) 9 20

a MTB, M. tuberculosis.b One (or a few) colonies growing on Lowenstein-Jensen medium. For the

composite of other mycobacterial species that were negative on microcolonyplates, the average time to positivity on conventional media was 29 days. Ifthe microcolony plate was positive, the average time to positivity was 9 days,and if the conventional medium was positive, the average time to positivitywas 19 days.

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FIG. 1. Microcolony morphologies of selected mycobacterialspecies on Middlebrook 7H11 agar. Photographs were taken as thecolonies were visualized for detection, through the bottom of theplastic petri dish and the 10-mm layer of Middlebrook 7H11 agarmedium, by bright-field microscopy at magnifications of x 110 tox 160, with colonies ranging in age from 3 to 12 days. (a) Colonies ofM. tuberculosis examined at x 110 magnification at 10 days of age.(b) Colonies ofM. tuberculosis examined at x 110 magnification and12 days of age. (c) Colonies of M. kansasii examined at x 110magnification and 10 days of age. (d) Colonies of MAI examined atx 125 magnification and 4 days of age. (e) Colonies of MAI examinedat x 160 magnification and 7 days of age. (f) Colonies ofM. gordonaeexamined at x 110 magnification and 10 days of age. (g) Colony ofM.fortuitum examined at x 110 magnification and 3 days of age. (h)Colonies of Nocardia (four filamentous colonies) and yeast (singleentire colony) growing on the medium showing the distinctivecolony morphologies of nonmycobacteria.

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TIMELY CULTURE FOR MYCOBACTERIA 2181

with less sensitivity by the microcolony method; in many of

F; those instances, the original acid-fast smear of the concen-trated specimen was negative. Although less sensitive than

f s1I the method with conventional media, the microcolony

method displayed an advantage in speed of isolation. Theaverage interval to detection, also depicted in Table 1, wasmarkedly reduced by the microcolony method. Overall, forM. tuberculosis the average time to detection was only 10days by the microcolony method, compared with 21 days bythe conventional method, and for MAI these figures were 11and 25 days, respectively. With the exception of M. gordo-nae, for which the reduction in time to detection was onlymodest, the microcolony method yielded detection of allmycobacterial species in substantially shorter time periodsthan were required by the conventional method.Because the microcolony method yielded such a substan-

tial improvement in detection time over the conventionalmethod, the data were further evaluated to determine howthe addition of another medium might restore the full sensi-tivity for detection of the most critically important mycobac-terial species. For M. tuberculosis and MAI, stratification ofculture results in relation to acid-fast stain results revealedthe findings shown in Table 2. The microcolony method wasnegative primarily in instances in which the acid-fast stain ofthe concentrated clinical specimen was negative. In suchinstances, M. tuberculosis was grown in small colony num-bers only on the Lowenstein-Jensen medium, and the aver-age time to first detection was 33 days. With the balance ofstain-negative specimens that were culture positive for M.tuberculosis, the time to first detection averaged 16 days forthe microcolony method and 24 days for the macroscopicmethod. In contrast, the time to positivity for stain-positivespecimens that were culture positive for M. tuberculosisaveraged 7 days for the microcolony method and 17 days forthe macroscopic method. Similar results were observed withother mycobacteria. Thus, for optimized culture of themycobacteria, a single plate of 7H11 medium observed formicrocolonies would provide an optimal time to first detec-tion and a single tube of Lowenstein-Jensen medium wouldprovide complementary coverage, with the composite re-

-,-.> -z;-d,-$ storing the culture protocol to full sensitivity. We hereafteruse the term microcolony system to refer to the composite of

h..#.A:<.v t = ............................one thin plate screened microscopically and one Lowen-,..gwe. 21 3 li l l1 |~ .................... stein-Jensen tube evaluated macroscopically.Figure 1 depicts the colonial morphologies of the myco-

bacterial colonies. To demonstrate that these are readilydifferentiable from other colony types, we include a photoshowing yeasts and a Nocardia sp. that were detected within3 days of incubation. Other artifacts that appear on the platesinclude patient cells, from undigested specimens, and occa-sional bacterial colonies, indigenous organisms that survivethe decontamination process and that resist the selectivepressures in the medium; the microscopist soon learns toappreciate other bacterial colonies for their distinctive char-acteristics without generally needing to perform acid-faststainings, much as in macroscopic evaluations. M. tubercu-losis yields colonies that exhibit cording. Even when thereare only a few cells in the microcolony, they appear as

~~ - - - ~~distinct cells arranged in rows. As the colony matures theE`-X _, margin is irregular and the cording appears to be quite

distinct. For the novice, an early colony of M. kansasii ischallenging to distinguish from M. tuberculosis, whereas anolder colony of M. kansasii is more readily differentiable.MAI has a colonial morphology that is quite distinct fromthat of M. tuberculosis. The colonies are smooth with a

margin that is entire. Colonies of MAT younger than 7 days

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may be either dense and compact or spreading. With time,MAI colonies tend to develop the appearance of a fried egg,with dense centers and a wide border or apron that is lessdense. With increasing experience it is possible to distin-guish the early colonial morphologies and achieve a pre-sumptive identification of the most commonly encounteredmycobacterial species. Solely on the basis of colonial mor-phology, presumptive identification could be made after an

average of 10 days of incubation for 83.5% of the cases of M.tuberculosis and by 11 days in 85% of the cases of MAI. Ina few cases, presumptive identification was deferred or wasinaccurate. Such presumptive identification was useful forthe selection of appropriate confirmatory identification path-ways and for verbal presumptive reports, but it was notsufficiently reliable for disclosure on written laboratoryreports.

DISCUSSION

The microcolony system, a composite of the microcolonymethod and a single tube of Lowenstein-Jensen medium, isboth rapid and sensitive for the recovery of mycobacteria inculture. For acid-fast stain-positive specimens, the detectionof M. tuberculosis requires an average of only 7 days; forstain-negative specimens, the average time to first detectionis 16 days, with an overall average of 10 days. Among thespecies of mycobacteria there are two subsets of colonytypes: the group with a colony type that bears a resemblanceto that of M. tuberculosis and the group with a colony typethat more closely resembles that of MAI by virtue of thedense centers, the fried egg appearance. Presumptive iden-tification of these two species on the basis of colonialmorphology may serve to guide the clinician regardingtherapy and infection control and definitely guides the labo-ratorian in the subsequent steps that need to be taken forconfirmatory identification. Colonial morphology directs theselection of a single gene probe for rapid culture confirma-tion of these two species, allowing the identity of theorganism to be confirmed within hours of detection of thecolony. Although not used for confirmatory identification forthe purposes of the present study, DNA hybridization hasbeen applied successfully on a limited basis. Signals 10-foldgreater than the cutoff can be generated with a colony bulkthat is not visible macroscopically. Since a single probe canbe selected, costs are reduced and this enhances the labora-tory's ability to cost-effectively turn out results withoutbatching tests. Given the rapid recovery and identificationtimes, a laboratory might provide better service to its clientsby using the microcolony culture system than it could bysending specimens to a reference laboratory for radiometricculture or genetic amplification technologies, because thedays lost in transit would compromise the overall speed offurnishing information. Moreover, some reference laborato-ries have a tendency to batch their tests, and this prolongsthe average turnaround time when specimens are receivedjust after a test series has been run.

Genetic amplification methods that are currently underevaluation give earlier results on some stain-negative speci-mens but are inadequately sensitive to detect mycobacteriaon the order of half of all stain-negative, culture-positivespecimens (6). Presumably, these are comparable to speci-mens that in this series yielded one or a few colonies andaveraged over 4 weeks for recovery. Thus, with all currentmethods the greatest challenge, early detection of organismsnot detected by an acid-fast staining, is still not met. Fortu-nately, in many of these cases, transmissibility is of dimin-

ished consequence because of the small numbers of organ-isms being shed. When limited to respiratory samples, somepolymerase chain reaction test protocols appear to have ahigh degree of sensitivity (3, 5, 8), but their application inhigh-prevalence populations has revealed a potential prob-lem with false-positive signals when testing blood and respi-ratory samples from case contacts and from individuals witha prior history of tuberculosis, even among those treatedsurgically over 30 years ago (9). It is hoped that there willsoon be widely available refinements in amplification tech-nologies that will sensitively and specifically detect theseagents and thus allow for more early diagnosis and institu-tion of therapy. The microcolony system is an ideal bridge tothat technology, because no capital investment is required; ituses a conventional microscope rather than a dissectingmicroscope, because the depth of the medium accommo-dates the working distance of a standard light microscope.The recent experience of other investigators has demon-

strated that the use of plated media in mycobacteriology mayhave advantages of speed over the radiometric method.Working with blood specimens, Evans et al. (4) have notedthat in over 50% of cultures, detection of mycobacteremia isearlier with Middlebrook 7H11 plates than by the radiomet-ric method. Although the time to detection by the microcol-ony method was not compared directly with that of theradiometric method in our evaluation, one might infer thatthe two methods would yield a comparable time to positiv-ity. The findings of another group (10) have documented areduction in the interval to first detection of 10 days whencomparing the radiometric method with conventional culturemedia. Similarly, in our investigation comparing the micro-colony method with conventional media, the average time todetection was reduced by 11 days for M. tuberculosis. Evenwith similar times to detection, however, the two methodsmay vary in applicability among different laboratory opera-tions. The microcolony method may be too labor-intensivefor a large laboratory but may be suitable for a mycobacte-riology laboratory of moderate size. When using an opti-mized microscopic setup and screening at x40 magnifica-tion, a proficient microscopist requires 20 s to screen the firsttwo quadrants of each plate for the appearance of microcol-onies. For a laboratory that receives 100 (or 200) cultures permonth, this means that 3.3 (or 6.6) h per month would bedevoted to this activity. With the microcolony method, asthe culture volume rises, the technologist time rises propor-tionately. In contrast, with the radiometric method severalof the routine activities are independent of culture volume,for example, preventive maintenance and radioisotope mon-itoring. Other activities bear some proportional or stepwiserelationship to culture volume, for example, discardingradioisotope if the bottles are opened and their contents arepoured down the sink, loading the trays onto the monitoringdevice, cleaning the probe, and dealing with false-positivesignals. Depending on local requirements for isotope man-agement and preventive maintenance, at some moderateculture volume the cumulative technologist time that wouldbe required for the instrumented method would be similar tothat required for the microcolony method. Other consider-ations may also play a role in the relative ease of adaptationto one of the two methods. For example, with the microcol-ony method the presumptive identity is immediately avail-able, whereas with the radiometric method, further subcul-ture and/or manipulation, either with the p-nitro-oa-acetylamino-o-hydroxypropiophenone test or a geneticprobe, is required. Moreover, there may be significantadvantages in biosafety for some laboratories working with

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the microcolony method. For example, inoculation of theplated and tubed media does not require the use of a needleto puncture the stopper as does the radiometric method.Furthermore, manipulation of positive bottles containinghigh levels of mycobacteria in fluid culture represents apotentially greater hazard for the accidental production ofaerosols than does the manipulation of the microcolonies.Such safety considerations may play a greater role in deci-sion making should multiply drug-resistant strains becomemore prevalent. Another important consideration may be thecost of evaluating the specimens. For laboratories withculture volumes such that the cumulative technologist timefor the two methods is equivalent, the microcolony methodbecomes substantially less expensive than the radiometricmethod. Thus, each laboratory should base its decision onwhich methodology that it should use on a thorough consid-eration of all relevant factors.The microcolony method possesses a number of potential

enhancements that were not directly studied in the presentevaluation. First, with acid-fast stain-positive specimens,the laboratory staff could inoculate in parallel additionalplates containing threshold levels of antimycobacterial drugsthat would allow them to screen rapidly and inexpensivelyfor resistance. Second, the microcolony plate provides dis-tinctive colonial morphology at first detection. This allowsfor the immediate selection of the appropriate genetic probe,eliminating the necessity of routinely applying more than oneprobe to provide confirmatory identification as is oftenencountered for the rapid identification of these mycobacte-ria when they are grown in broth systems such as theBACTEC radiometric system. Third, the distinctive colonialmorphology also allows for the rapid recognition of mixedmycobacterial infections. Because mixed mycobacterial in-fections are noted increasingly in certain populations, forexample, in patients with AIDS, the method allows theclinician to provide early empiric coverage for dual infectingagents. With the radiometric method, such dual infectionsmay be missed or delayed until subcultures on solid mediaare available.The system described here, a composite of a single plate of

Middlebrook 7H11 medium, examined for microcolonies,and a single tube of Lowenstein-Jensen medium, examinedmacroscopically, provides a timely culture result that mayhave advantages for many laboratories, pending the success-ful introduction of genetic amplification methodologies. Withtechnologist time at a premium, it is wise to minimize thetime spent evaluating these cultures. On the basis of a reviewof the time of detection of mycobacteria on the two media, itis possible to devise a culture examination schedule thatwould optimize for early detection and full sensitivity whilefacilitating the scheduling of staff time effectively. Themicrocolony method yielded earlier detection in 84 of 85 ofthe cultures in which both the 7H11 medium and the Lowen-stein-Jensen medium were positive. In the one aberrantculture, the Lowenstein-Jensen medium was positive on day18 of incubation and the 7H11 medium was positive on day20 of incubation. In the 18 cultures in which only theLowenstein-Jensen medium was positive, the time to firstdetection for all cultures was on or after day 21 of incuba-tion. Since macroscopic detection of colonies on Lowen-stein-Jensen medium is slower than microscopic detectionon the microcolony plate, it would not be necessary toexamine the Lowenstein-Jensen medium early in the courseof its incubation. A rationalized culture evaluation protocolmight consist of twice (or thrice)-weekly evaluation of themicrocolony plates for the first 3 weeks, with a terminal

review of the plate at the end of the fourth week. During thefourth week of incubation, the speed and efficiency ofrecovery of M. tuberculosis on Lowenstein-Jensen mediumbecomes equivalent to that of the microplate. Thus, it doesnot appear that it would be beneficial to extend the incuba-tion of the microplate beyond this period. The Lowenstein-Jensen medium could be evaluated twice weekly, beginningwith day 21 of incubation and continuing through day 35 ofincubation, and with evaluation thereafter being onceweekly through the eighth week. By retrospectively model-ing the culture protocol used in the present study, it wasdetermined that delaying examination of Lowenstein-Jensenmedium until day 21 of incubation would have eliminated72,000 tube examinations and delayed a single positiveculture result by 2 days. If delay in initiating reading of theLowenstein-Jensen medium until day 21 would allow thetechnologist to increase the frequency of reading of themicrocolony plates from twice to thrice per week, it woulddoubtless promote the earlier detection ofM. tuberculosis ina number of additional cultures. Mutually exclusive reviewperiods for the two media are an efficient means of facilitat-ing scheduling of personnel in the mycobacterial culturelaboratory. Because of the requirement for multiple speci-mens for individual patients and the variable ordering prac-tices among physicians, tuberculosis culture volumes mayvary considerably from month to month within a givenlaboratory. Large increases in work load may tax the capa-bility of the laboratory staff. With such a culture evaluationprotocol, the complementary medium would require addi-tional technologist time only after completion of the inten-sive review period required for the microcolony plates. Thesurge that occurs in one month is partially delayed until thenext month if half of the culture tubes do not requireexamination for the first 3 weeks.As with all methods, the microcolony method described

here is only as adequate as the specimen and the affiliatedactivities. The laboratory should seek to optimize the trans-port, processing, and reporting process in order to minimizethe response time required for delivery of effective therapyand for institution of infection control measures. The labo-ratory must review all of its specimen and test parameters toproduce an optimal test outcome, the standard for which hasrecently been specified (1). Clinical specimens should reachthe laboratory within 24 h of collection; this should befollowed within another 24 h by the issuance of smearreports. Positive cultures should be detected within 14 daysof specimen receipt, with identification and susceptibilitytest results rendered within 21 and 28 days, respectively.Conformance to these standards will contribute significantlyto the containment of tuberculosis and reduce the threat ofmultiply drug-resistant tuberculosis.

REFERENCES

1. Centers for Disease Control. 1992. National action plan tocombat multidrug resistant tuberculosis. Recommendations andreports. Morbid. Mortal. Weekly Rep. 41(RR-11):13-16.

2. Daniel, T. M. 1990. The rapid diagnosis of tuberculosis: aselective review. J. Lab. Clin. Med. 116:277-282.

3. Eisenach, K. D., M. D. Sifford, M. D. Caves, J. H. Bates, andJ. T. Crawford. 1991. Detection ofMycobacterium tuberculosisin sputum samples using a polymerase chain reaction. Am. Rev.Respir. Dis. 144:1160-1163.

4. Evans, K., D. Forthal, and E. M. Peterson. 1992. Detection ofacid fast bacteria from blood cultures, abstr. U-37, p. 171.Abstr. 92nd Gen. Meet. Am. Soc. Microbiol. 1992. AmericanSociety for Microbiology, Washington, D.C.

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5. Forbes, B. A., and K. E. Hicks. 1992. Detection of Mycobacte-rium tuberculosis in respiratory specimens in a clinical labora-tory setting using the polymerase chain reaction, abstr. 18B.World Congress on Tuberculosis. National Institutes of Health,Washington, D.C.

6. Nolte, F., B. Metchock, A. Edwards, 0. Okwumabua, and T.Shinnick. 1992. Direct detection of Mycobacterium tuberculosisin sputum using the polymerase chain reaction, abstr. U-1, p.165. Abstr. 92nd Gen. Meet. Am. Soc. Microbiol. 1992. Amer-ican Society for Microbiology, Washington, D.C.

7. Runyon, E. H. 1970. Identification of mycobacterial pathogensutilizing colony characteristics. Am. J. Clin. Pathol. 54:578-586.

8. Savnc, B., U. Sjobring, S. Alugupalli, L. Larsson, and H.Miorner. 1992. Evaluation of polymerase chain reaction, tuber-culostearic acid-analysis and direct microscopy for the detection

of Mycobacterium tuberculosis in sputum. J. Infect. Dis. 166:1177-1180.

9. Shaw, R. J., D. Walker, I. K. Taylor, and D. M. Mitchell. 1992.Clinical study of polymerase chain reaction amplification ofIS6110 DNA in respiratory and blood samples in the diagnosisof active tuberculosis, abstr. 20C. World Congress on Tubercu-losis. National Institutes of Health, Washington, D.C.

10. Stager, C. E., J. P. Libonati, S. H. Siddiqi, J. R. Davis, N. M.Hooper, J. F. Baker, and M. E. Carter. 1991. Role of solid mediawhen used in conjunction with the BACTEC system for myco-bacterial isolation and identification. J. Clin. Microbiol. 29:154-157.

11. Vestal, A. L., and G. P. Kubica. 1965. Differential colonialcharacteristics of mycobacteria on Middlebrook and Cohn 7H10agar-base medium. Am. Rev. Respir. Dis. 94:247-252.

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