JOURNALOFCLINICALMICROBIOLOGY, Aug. 1983,p.384-388
0095-1137/83/080384-05$02.00/0
Copyright C 1983, AmericanSocietyforMicrobiology
Vol.18, No. 2
Comparison
of
a
Radiometric
Method
(BACTEC)
and
Conventional Culture
Media
for
Recovery
of
Mycobacteria
from
Smear-Negative Specimens
MARGIE A.MORGAN, CARL D. HORSTMEIER,DONALD R. DEYOUNG, AND GLENN D. ROBERTS*
MayoClinic and Mayo Foundation, Rochester, Minnesota 55905
Received 29 November1982/Accepted 18 April 1983
The BACTEC system and three conventional media (Middlebrook 7H10,
selective Middlebrook 7H11 [S7H11], and Lowenstein-Jensen [LJ]) were
com-pared for their mean recovery times and recovery rates of mycobacteria from
acid-fast, smear-negative clinical specimens. Of the 71 smear-negative,
culture-positive specimens recovered from 2,165 submitted smear-negative cultures, the
BACTEC system detected 71.8%, compared with 88.7% for the conventional
three-mediumsystem.When mediawereindividually compared,BACTEC
medi-um(Middlebrook7H12)wasmoresuccessful in recovering mycobacteria (71.8%)
thanwas LJ (62%), Middlebrook medium 7H10(55.9%), orMiddlebrook S7H11
medium(52.1%). Middlebrook 7H11 mediumcontaining sodium selenatewas also
evaluated and didnotincreasethe recovery rate or decrease the recovery time of
mycobacterial species when comparedwith LJ, Middlebrook 7H10, S7H11, and
7H12media. Themeandetection time for theBACTEC systemwasless than that
by conventional methods for the seven species of mycobacteria recovered.
Detection times for Mycobacterium tuberculosis on the BACTEC system and
conventional cultural systems were13.7 and 26.3 days, respectively.
The BACTEC system (Johnston
Labora-tories, Inc.,Cockeysville, Md.)hasbeen
report-ed to be valuable for the rapid detection of
clinically important mycobacteria. The system
uses Middlebrook7H12 broth medium
contain-ing14C-labeled palmitic acidfortheradiometric
detection ofmycobacterialgrowth and has been
showntobemoresuccessful indetecting
myco-bacteria than conventional methods involving
only one medium (C. D. Horstmeier, D. R.
DeYoung, K. A. Doerr, and G. D. Roberts,
Abstr. Annu. Meet. Am. Soc. Microbiol. 1982.
C187,p. 302).
Previous studies reported the BACTEC
sys-tem tobe assuccessfulasconventionalculturing methodsin detecting growth in specimens
hav-ing apositive direct smearfor acid-fast bacilli.
These studies also revealed that the BACTEC
system detected mycobacterial growth more
rapidly than did conventional cultural methods
(Horstmeier et al., Abstr. Annu. Meet. ASM,
1982,C187;N.Goodman,H.Larsh,T.Lindner, M. McGinnis, K. McClatchy, and G. Roberts, Abstr. Intersci. Conf. Antimicrob. Agents Che-mother. 21st,Chicago, Ill., abstr.no.96,1981).
It has been reported that detection times for
mycobacterial growth in acid-fast
smear-posi-tive specimens, as detected by the BACTEC
system, varied among the five medical centers
that evaluatedthe method, witha meanof8.36
and 5.4 days for Mycobacterium tuberculosis
and mycobacteria other than tubercle bacilli
(MOTT), respectively (Goodman et al., 21st
ICAAC, abstr.no.96).Meandetectiontimes for
M. tuberculosis and MOTT were 13.2and22.8
days, respectively, as determined by
conven-tional methods.
A specimen with a negative acid-fast smear
and apositive cultureformycobacteriaisnot an
unusual occurrence inaclinicallaboratory.The present study evaluated the BACTEC system
and conventional methods for detecting
myco-bacteria in specimens with a negative acid-fast
smearandapositiveculture. Middlebrook7H12
(BACTEC) mediumwascompared with
Lowen-stein-Jensen (LJ), Middlebrook 7H10, and
Middlebrook S7H11 mediafor comparative
re-covery rates and times of seven species of
mycobacteria foundinclinical specimens.
Mid-dlebrook 7H11 containing sodium selenate was
also compared with LJ, Middlebrook 7H12,
7H10, and 7H11 for the recovery times ofM.
384
on February 8, 2020 by guest
http://jcm.asm.org/
MYCOBACTERIA RECOVERY BY BACTEC AND OTHER MEDIA 385
BACTEC | =71.8%
0) 0)
CONVENTIONAL =88.7%
Q
0D
FIG. 1. Comparison of the number of
mycobacte-ria(excludingM. gordonae)recovered from 71 acid-fast, smear-negative specimensbythe BACTEC and conventional methods.
tuberculosis and MOTT. Sodium selenate was
previously reportedto enhance thegrowthof M.
tuberculosis (1).
MATERIALSANDMETHODS
Acid-fast smear-negative specimens, excluding
ce-rebrospinal fluid and urine, submittedto our labora-toryduring a3-month period (June toAugust, 1981)
for routinemycobacterialculturewereincluded inthis
study. Of 2,165 specimens, 71 had negative direct acid-fast smears, butmycobacteriawererecoveredby
either the conventional or the radiometric method.
Specimensfrom whichMycobacteriumgordonaewas
recoveredwere notincluded in this group. Tissue and body fluidsprocessed for mycobacterial culturewere
inoculated directly onto culture media. Respiratory
tract specimens andgastric washingswereprocessed by using2% NaOH decontamination and
centrifuga-tion (3). The BACTEC method used liquid Middle-brook 7H12(Johnston Laboratories, Inc.)containing
"C-labeled palmitic acid. The conventional cultural
technique used three media: Middlebrook 7H10, Middlebrook S7H11 containing polymyxin B (20
,ug/ml), trimethoprimlactate (20,g/ml), and carbeni-cillin (50 ,ug/ml), and two LJ slants (Difco Labora-tories). Selenate 7H11 containing 5 ,g of sodium selenate per ml (Diagnostics, Inc., St. Paul, Minn.) was also evaluated in this study. The Middlebrook 7H10, 7H11, and S7H11 mediawereinoculated with 0.5 mlof concentrated specimen, and twoLJ slants
wereinoculatedwith 0.25 ml each. The 7H12 medium wasinoculated with 0.1 ml of concentrated specimen according to the manufacturer's instructions. A small amountof sediment from the concentratedspecimens was used to prepare smears forauramine-rhodamine staining. The petri dishes containing 7H10, S7H11,
andselenate 7H11 media were sealed inpolyethylene, C02-permeable plastic bags, and allmedia, including LJslants, were incubated for 8 weeksat35C in an
atmosphere of 5 to 10% CO2. Conventional media wereexamined everythirdday for visibleevidence of growth until week 4ofincubation, when they were examinedonceweekly.BACTEC bottleswerereadby using a BACTEC model 460 every third day until week 4ofincubation; then theywerereadonceweeklyfora
totalof 8 weeks. BACTEC bottles withagrowth index reading of -10 were considered to bepositive. Posi-tivebottles were read daily until areading of 100was
reached, and then samples of each medium were used to prepare acid-fast stains and subcultures on LJ medium. Subcultures were incubated at 35°C in an atmosphere of 5 to 10%oC02 until growth was suffi-cientfor biochemical testing.Mycobacteriawere iden-tifiedby conventional methods (2, 3).
RESULTS
A total of 134 mycobacteria were recovered
from2,165 acid-fast, smear-negative specimens
submitted for culture. Sixty-threeisolates of M. gordonae were included; however, these were notconsidered in theanalysisof the data. Bacte-rial contamination appeared in 78 of 2,165
(3.6%) specimens cultured for mycobacteria.
Whencontamination occurred in the 7H12 broth
medium,the broth wassubculturedonto ablood
agar plate and 7H10 medium. Mycobacteria
were recovered from three contaminated
speci-mens from the smear-negative group. Six cul-tures from threepatients yielded morethan one
organism. M. gordonae was the second orga-nism in allexcept two cultures inwhich
Myco-bacteriumavium-intracellulare and
Mycobacte-riumfortuitum were recovered simultaneously.
Sevenspecies of mycobacteria, excluding M.
gordonae, were recovered from the 71
smear-Most Rapid Recovery Time On Conventional Media (Days)
<7 8-14 15-21 22+
<783 1 1
MostRapid Recovery Time
InBactec Medium(Days)
8-14
15-21
...
...
...
... ... ~~~~~~~~.O~~~~~~~~~~~~~~~~~...Slower Bacte .a eTm
FIG. 2. Comparisonof recovery times for mycobacteriafrom acid-fast, smear-negativespecimens, using the BACTEC and conventional methods.
VOL.18,1983
on February 8, 2020 by guest
http://jcm.asm.org/
386 MORGAN ET AL.
Organism:
M.avium
M.tuberculosis
M.fortuitum
M.chelonei
8. 1 MEAN TIME
2 Lt ... ... ... ::.RANGE::: 35 -I BACTEC ME
I
...2...
12 _I_AI0 28
4.0
4
13.7 20.1
16L 147
26.3
3
396.5
4 1:;A -I8:: 22.4
] 37 18.0
.THOD
MEAN TIME
(DAYS) SAVED
BY BACTEC METHOD18.3
Mavium 1 __: 46
M.tuberculosis 1 16 19 '2isolates 5 and 12
M.fortuitum 9 9 days longer
M.chelonei 2 __I 31
I I11.8 1 1 1 1
0 DAYS 50
Organism:
11.0 MEAN TIMEM.gastri
g.
.i
g
RANGE
+-
BACTEC
METHOD
39 46
17.0 42.5
M.kansasii
::
17'
17J35
27
13.3 31.0
M.gordonae
|91t125J,
MEAN
TIME (DAYS) SAVED BY BACTEC
38.8O
M.gastri
,3
M.kansasii 19
M.gordonae 4 l 116
I . .
....
I.0 DAYS 50
FIG. 3. Distribution of recovery times of mycobacteria from acid-fast, smear-negative cultures, using the BACTEC and conventional methods.
negative specimens. M. avium-intracellulare
was the mostcommonly recovered (37 cultures),
followed in decreasing order ofoccurrence by
M. tuberculosis (14cultures), M.fortuitum (10
cultures), Mycobacterium chelonei (6cultures),
Mycobacterium kansasii (2 cultures), and
Myco-bacteriumgastri (2cultures).
Ofthe 71 positive mycobacteria cultures, the
conventional method detected 63 (88.7%) and
theBACTEC method detected51 (71.8%) (Fig.
1). TheBACTEC system wasfoundtobe more
rapidindetecting41 of the 43 cultures
eventual-ly detected by both systems (Fig. 2). The
re-mainingtwocultures were detected on the same
day by bothsystems.
Theranges and mean recoverytimes(in days)
for seven species of mycobacteria were
deter-mined for the BACTEC system and
convention-altechniques. The BACTEC system was more
rapid than the conventional system for the
de-tectionof all seven mycobacterial species (Fig.
3). The mean time saved bythe BACTEC
sys-tem depended on the species of mycobacteria
recovered but was more than that savedbythe
conventional system for all species evaluated
(Fig. 3and4).
The species ofmycobacteriarecovered from
the 71 acid-fast, smear-negative specimens by
conventional methods and not recoveredby the
BACTEC system were determined and
includ-ed: M. avium-intracellulare(5cultures),M.
for-tuitum (8cultures),M.chelonei(3cultures),M.
J.CLIN. MICROBIOL.
I
on February 8, 2020 by guest
http://jcm.asm.org/
TABLE 1. Comparison of media for the recovery of
mycobacteriafrom acid-fast, smear-negative
specimens(n =71)
No. of Mycobacteria recovered in:
specimens BACTEC
LU
7H10 7Hll21 + + + +
4 + + + _
6 + + - +
5 + + - _
5 + - + +
2 + - + _
8 + - _ _
9 - + _ _
1 - + - +
2 - + + _
1 - - + +
4 - - + _
3 - - - +
No. recovered 51 44 39 37 % Recovered 71.8 62.0 54.9 52.1
tuberculosis(2 cultures), M. kansasii (1 culture), and M. gastri(1 culture). Conventional cultural methods failed to recover the following
myco-bacteria: M. avium-intracellulare (6 cultures), M.fortuitum (1 culture), and M. tuberculosis (1 culture).
In the evaluation of 71 specimens, Middle-brook 7H12 medium was more successful in recovery of mycobacteria than was any one
solid medium (Table 1). The BACTEC system
detected 71.8% of allmycobacteria, whereasLJ,
7H10, andS7H11 detected62, 55.9, and 52.1%, respectively.
Growthof M. tuberculosiswasnot significant-ly enhanced by theaddition of sodium selenate to 7H11 medium. The mean recovery times of M. tuberculosis and MOTTas determined with
Middlebrook 7H12,LJ,7H10, S7H11,and sele-nate 7H11 were analyzed. The recovery times
for M. tuberculosisandMOTTweredetermined
for thefive media. All fivemedia recovered the
mycobacteria on approximately the same day.
Therefore, studies indicated that selenate7H11
didnotdecrease the meanrecovery time of M.
tuberculosis or ofMOTT compared with other media studied.
DISCUSSION
This evaluation compared the BACTEC
sys-temwithconventionalculturing methods for the detection ofmycobacteria from acid-fast,
smear-negative, culture-positive specimens. The growth of mycobacteria from an acid-fast,
smear-negative specimen is the first clinical indi-cation of a possible mycobacterial infection.
Becauseapproximately 40% of the positive
my-cobacterial cultures inourlaboratory have
nega-tive direct acid-fast smears, the ability of the
BACTECsystem todetectmycobacteria rapidly
in thistype of specimen issignificant.
M.gordonae was eliminated from most
evalu-ations, and onlyclinically relevantisolates were
considered. This highrecovery rate ofM.
gor-donae in our laboratory, however, does present
problems with the BACTECsystem.Currently,
the BACTEC system fails to provide isolated
colonies and has no method to identify the
species of mycobacteria detected. The
suspen-sion of mycobacteria must be cultured onto
conventional media beforeabiochemical
identi-fication can be made. A culture medium was
developed containing NAP (p-nitro-a-acetyl-amino-,B-hydroxy-propiophenone), which aids in
separatingM. tuberculosis from MOTT but will
notidentifyM. gordonae(A. Laszlo and S. H.
Siddiqi, Abstr. Annu. Meet. Am. Soc.
Microbi-ol. 1982,C189, p. 303).
The BACTEC system detected 71.8% of
smear-negative mycobacteria cultures,
com-pared with 88.7% detected by the conventional
system.The latter used atotalvolume of 1.5 ml
of concentrated specimen on twoculture slants
and two agar plates, whereas the BACTEC systemusedonly0.1 ml of concentrated
speci-men to inoculate one bottle containing 2 ml of liquid 7H12 medium. The increased specimen
volume used to inoculate the fourmedia could
account for the increased detection rate
achieved by the conventionalmethod.Our
labo-ratory is presently evaluating the usage of
in-creasedinoculum size inthe Middlebrook7H12
medium to possibly increase the sensitivity of
mycobacterial detection. However, the
sensitiv-ity of the 7H12 medium for recovering
mycobac-teria exceeded that of any individual
conven-tional medium evaluated alone (Table 1). The
7H12medium recovered71.8% of the
mycobac-teria, compared with the recovery rates for LJ
(62%), 7H10(55.9%), and S7H11 (52.1%).
Sodium selenate incorporated into 7H11 agar
has beenreported toenhance the growth ofM.
tuberculosis (1).In ourlaboratory, sodium
sele-nate addition to 7H11 medium did not
signifi-cantly decrease therecoverytime ofM.
tubercu-losisorMOTTcomparedwith LJ,7H10,S7H11,
or7H12orenhance the recovery rate.
In this study, the BACTEC system proved
more rapid for the detection of mycobacteria than were conventional culture methods. The
detection of mycobacteria is helpful, but the
institution of chemotherapy depends on the
identification of the species of organism
recov-ered from the clinical specimen. The necessity
exists for either rapid identification or rapid
susceptibility testing. Techniques involving the
BACTEC system areavailablefor rapid
suscep-tibility testing. Results ofcollaborative studies
suggest that drugsusceptibility testing data can
VOL.18,1983 387
on February 8, 2020 by guest
http://jcm.asm.org/
388 MORGAN ET AL.
be obtained 4.2 to 6.9 days after detection of
mycobacterial growth; however,acorrelatecan
only be made by both methods forM.
tuberculo-sis susceptibility testing (Goodman et al., 21st
ICAAC, abstr. no.96).
Themeanrecoverytimeformycobacteriawas
decreased with the BACTEC system as
com-pared with theconventional system. However,
itmustbenoted that the BACTECsystemfailed
to detect 20 positive specimens, as compared
with8 missed by theconventional system. The
BACTEC system had particular problems in
detecting specimens yielding M.fortuitum and
M. avium-intracellulare. The conventional
sys-temshared the lowrecovery rate ofM.
avium-intracellulare butwas moresuccessful in
recov-J. CLIN.MICROBIOL.
eringM.fortuitum. Thereasonfor thisrecovery
discrepancyis notknown; perhaps the
antibiot-ics in the medium wereinhibitory.
Datafrom this and other studies indicate that
BACTEC offers promise as a method for the
rapid detection of mycobacterial growth. Atthis
time, however, this systemshould be used
con-currently with conventionalculturingmethods.
LITERATURE CITED
1. Jacquess, P. A., D. L. Smalley, and J. K. Duckworth. 1981. Enhanced growth ofMycobacterium tuberculosis in the presenceof selenium. Am. J.Clin.Pathol. 75:209-210. 2. Kubica, G. P. 1973. Identification of mycobacteria. Am.
Rev.Respir.Dis. 107:9-21.
3. Roberts, G. D. 1982.Mycobacteria and Nocardia, p. 365-406. InJ. A.WashingtonII(ed.), Laboratory procedures in clinical microbiology. Springer-Verlag, New York.
on February 8, 2020 by guest
http://jcm.asm.org/