Tuberculosis: Pathogenesis, Protection, and Control
Edited by Barry R. Bloom
© 1994 American Society for Microbiology, Washington, DC 20005
Chapter 6
Cultivation of Mycobacterium tuberculosis for
Research Purposes
Lawrence G. Wayne
Individual investigators have different rea­
sons for cultivating Mycobacterium tuber­
culosis in their laboratories; the medium to
be used and the conditions of incubation
should be tailored to the specific goals,
taking into account the physiologic charac­
teristics of this organism. The most com­
mon reason for cultivating tubercle bacilli is
to confirm a clinical diagnosis of tuberculo­
sis. Procedures for isolating M. tuberculo­
sis from clinical specimens are well docu­
mented in many good clinical laboratory
manuals, and that aspect of their cultivation
will not be discussed further in this review.
I shall concentrate instead on those aspects
of the growth characteristics of M. tuber­
culosis that may influence the methods
used to cultivate the organism in a research
setting. Other species of mycobacteria will
not be considered except in passing or by
contrast, although the comments will usu­
ally be applicable to other members of the
M. tuberculosis complex, which encom­
passes M. tuberculosis, M. bovis (including
bacillus Calmette-Guerin [BCG]), M. afri-
canum, and M. microti (Wayne and Ku­
bica, 1986).
Lawrence G. Wayne • Tuberculosis Research Labo­
ratory (151), Department of Veterans Affairs Medical
Center, 5901 East S e v e n t h Street, L o n g B e a c h , Cali­
fornia 90822.
73
Among the nondiagnostic activities that
require cultivation of tubercle bacilli are the
following: (i) the study of dynamics of
growth and survival and the in vitro study
of drug susceptibility and mechanisms of
action, which may employ growth calcula­
tions based on colorimetric or turbidimetric
methods and/or actual plating and counting
of colonies; (ii) preparation of whole bacilli
for immunology or the study of pathogene­
sis; and (iii) preparation of bacillary prod­
ucts for immunologic, chemical, physio­
logic, or genetic study.
This chapter is not a cookbook of all
possible media or methods but instead is
intended to point the reader in the right
direction within a very large body of in­
formation, much of which was developed
during a "golden a g e " of tuberculosis re­
search, i.e., the early days of chemother­
apy, when the study of tuberculosis had not
yet fallen from favor and when lung associ­
ations and their journals still had the word
tuberculosis in their titles.
GENERAL CHARACTERISTICS OF
M. TUBERCULOSIS
Because things change slowly in myco-
bacteriology, the reader will find informa­
tion that is still relevant and useful in The
74 Wayne
Mycobacteria—a Sourcebook, a compre­
hensive sourcebook on mycobacteriology
that goes into great detail on the physiol­
ogy, chemistry, immunology, and pathoge­
nicity of this genus and encompasses the
state of the art up to 1984 (Kubica and
Wayne, 1984). The present review is of
narrower scope, expanding on those char­
acteristics that influence the selection of
methods and conditions to be used for
growing M. tuberculosis in the research
or applied biotechnology laboratory. The
most important point to be made here is
that the physiology, antigenicity, pathoge­
nicity, and chemical composition of tuber­
cle bacilli are all influenced by the com­
position of the culture medium and the
conditions under which the organisms are
grown. If results from different experiments
within a given laboratory or from experi­
ments conducted in different laboratories
are to be compared, it is essential that these
parameters be comparable or at least de­
fined.
A classic description of M. tuberculosis,
which would be only partially accurate,
might characterize this organism as a fas­
tidious, slowly growing, strictly aerobic,
lipid-rich, hydrophobic, acid-fast bacterial
rod.
It is true that tubercle bacilli are best
isolated from clinical specimens on rich and
fairly complex media, but the apparent fas­
tidiousness of some such isolates may be a
consequence of their injury by conditions in
host tissue or by the treatment employed
for processing the clinical specimen. Once
isolated, M. tuberculosis is capable of
adapting to growth on extremely simple
medium, i.e., one containing a simple
source of carbon and nitrogen plus some
buffer salts and trace elements. The func­
tions of various ingredients of simple and
complex media will be further discussed
below in the section on culture media.
It is true that tubercle bacilli have a long
generation time compared to that of most
commonly studied bacteria. Under optimal
conditions, M. tuberculosis requires 16 to
18 h to undergo one cycle of replication
(Wayne, 1977). With a generation time in
that range, a single bacillus can yield a
visible colony on solid medium within 2
weeks or less after inoculation. The exces­
sively long time of 6 to 10 weeks required
for detection of colonies on media planted
with some clinical specimens (Krasnow and
Wayne, 1969) is, again, probably the result
of a need to repair injury of the bacilli in the
specimen, as suggested above.
It is true that tubercle bacilli grow most
rapidly when well aerated and do not ap­
pear to multiply under completely anaero­
bic conditions. The overwhelming bulk of
research done on the chemistry and physi­
ology of M. tuberculosis has been con­
ducted with bacilli grown as pellicles or as
agitated dispersed cultures. However, the
choice of highly aerobic growth conditions
was usually dictated by a desire for rapid
production of good crops of cells without
an adequate appreciation of the physiologic
characteristics associated with a particular
degree of aeration. As Segal has noted in
his review of the physiology of tubercle
bacilli grown under different conditions
(Segal, 1984), M. tuberculosis has many of
the enzymes required for anaerobic metab­
olism, and indeed, the virulence of this
organism appears to be in part a function of
its ability to survive and/or grow under the
wide range of variation in partial 0 pres­
2
sures that may occur in healthy, inflamed,
or necrotic tissue. Furthermore, there are
marked differences in biochemical, anti­
genic, and pathogenic behaviors between
tubercle bacilli grown in vitro and those
grown in host tissues (Segal, 1984).
It is true that tubercle bacilli, like all
other mycobacteria, are very rich in lipid.
Furthermore, unlike some other mycobac­
terial species (Barrow et al., 1980), M.
tuberculosis does not produce a hydrophilic
outer sheath; it is indeed hydrophobic and
grows as a waxy pellicle in unagitated me­
dium and with extensive clumping in stirred

culture unless a detergent is included in the
medium. The tendency for pellicle forma­
tion reinforced the perception of this organ­
ism as a strict aerobe. However, except in
the early veil-like stage of surface growth,
the thick clumps that form in a pellicle or
agitated culture appear to enclose bacilli in
various degrees of 0 deprivation, and the
2 diminution of virulence for the experimen­
bacilli within a single culture undoubtedly
represent a physiologically heterogeneous
population (Wayne and Sramek, 1979).
MEDIA
Liquid Media
Much of the early research on the tuber­
cle bacillus was directed toward production
of tuberculin or other crude chemical prod­
ucts of the bacilli, such as unique lipids or
polysaccharides. Media containing very
simple and defined ingredients were devel­
oped to allow recovery of bacillary prod­
ucts free of macromolecular medium com­
ponents and to yield very high yields of the
slowly growing bacilli and their products.
These media, including those attributed to
Sauton, Long, Kirchner, Wong, and
Proskauer and Beck, were quite similar to
one another in composition. All contained
phosphate salts, citrate, M g S 0 , glycerol,
4 and 20 g of glycerol. Each ingredient must
and either asparagine or an ammonium salt
as nitrogen source (Soltys, 1952). Most
were supplemented with iron, and all con­
tained unrecognized trace minerals as im­
purities in the defined ingredients; these
trace elements were essential to satisfac­
tory growth (Affronti et al., 1990; Baisden,
1951). The glycerol was a carbon source
considered essential for copious growth as
a pellicle or as clumps in agitated medium.
As will be discussed below, the presence of
glycerol may have directed the metabolism
into a pathway that is not representative of
the physiologic state of the bacilli as they
occur in tissues of the infected host and
thus may have been counterproductive in
Chapter 6 • Cultivation of M. tuberculosis 75
the in vitro study of M. tuberculosis as it
may occur in tissues.
One specialized use of this type of me­
dium is for maintenance of the virulence of
a strain of M. tuberculosis. It has been
reported that cultivation of this organism in
detergent-containing medium results in
tal mouse. Bacilli obtained from a thin veil
of early pellicle growth from a detergent-
free synthetic medium, on the other hand,
are reported to be more virulent; when the
pellicle thickens and becomes bumpy with
age, virulence is again diminished (You-
mans, 1979, p. 332). Collins and colleagues
have presented evidence that the ages and
physiologic states of dispersed cultures in­
fluence the distribution between lung and
spleen of tubercle bacilli inoculated intra­
venously into mice (Collins et al., 1974).
For methods of preparing well-dispersed
bacilli for inoculation of mice, see the sec­
tion on inocula below.
The formulation for a representative ex­
ample of a simple synthetic medium for
cultivation of M. tuberculosis, the modified
Proskauer and Beck medium recommended
by Youmans (Youmans, 1979, p. 25), fol­
lows. Into 1 liter of glass distilled water are
dissolved, in the sequence listed, 5 g of
asparagine, 5 g of K H P 0 , 5 g of K S 0 ,
2 4 2 4
be dissolved before the next ingredient is
added. The pH is then adjusted to 6.8 to 7.0
with 40% NaOH, after which 1.5 g of mag­
nesium citrate is added, and the medium is
dispensed as needed and autoclaved at 15 lb
for 20 min. The asparagine or glycerol used
should not be of the highest purity, as trace
metal contaminants have been reported to
be necessary to support growth of M. tu­
berculosis (Affronti et al., 1990; Baisden,
1951; Youmans, 1979). On the other hand,
traces of fatty acids or heavy metals may be
inhibitory if they are introduced from inad­
equately cleaned or some dry sterilized
glassware. If the presence of albumin does
not interfere with the use for which the
76 Wayne
medium is intended, introduction of a ster­
ile stock solution to yield a final concentra­
tion of 0.5% will result in binding of traces
of toxic materials, permitting the bacilli to
grow.
The growth of tubercle bacilli in the syn­
thetic media cited is difficult to follow in
quantitative terms, since it cannot be mea­
sured optically and since simple plating is
not accurate because of the severe clump­
ing of the bacilli. This problem was solved
by the introduction of detergents into cul­
ture medium by Dubos and colleagues dur­
ing the 1940s (Dubos, 1945; Dubos and
Davis, 1946; Dubos and Middlebrook,
1948). Dubos liquid medium contains (per
liter) asparagine, 2 g; Casitone (Difco), 0.5
g; N a H P 0 , 2.5 g; K H P 0 , 1 g; ferric
2 4 2 4
ammonium citrate, 50 mg; M g S 0 , 10 mg;
4 The disadvantage of using a Tween 80-
CaCl , 0.5 mg; Z n S 0 , 0.1 mg; C u S 0 , 0.1
2 4 4 albumin medium is that the albumin supple­
mg; Tween 80, 0.2 g; bovine albumin frac­
tion V, 5 g; and glucose, 7.5 g, at a final pH
of 6.6 ± 0.2. To prepare a liter of the
medium, all ingredients except the albumin
and glucose are dissolved in 900 ml of
distilled water, and this basal medium is
autoclaved. The albumin fraction V and the
glucose are dissolved in 100 ml of saline and
filter sterilized; this supplement is added to
the basal medium aseptically, and the com­
plete medium is distributed to appropriate
sterile culture containers. In practice, de­
hydrated basal medium (Dubos broth base)
and sterile glucose-albumin supplement are
available commercially to facilitate prepa­
ration of this medium.
The most commonly used detergent,
Tween 80, is a polyoxyethylene derivative
of sorbitan monooleate, and it permits
growth of M. tuberculosis in liquid medium
in a diffuse form, as single cells or small
clusters of cells that are easily dispersed by
agitation. One problem associated with the
use of Tween 80 is that traces of free oleic
acid are released into the medium as a
result of either spontaneous hydrolysis or
specific enzymatic action of some species
of mycobacteria (Davis and Dubos, 1948).
Free oleate is toxic to M. tuberculosis, but
the problem is gotten around by adding
bovine serum albumin to the medium (Du­
bos, 1950). The oleate is complexed to the
albumin and thus is no longer inhibitory to
the bacilli; the oleate complexed to the
albumin can, however, be metabolized by
the bacilli (Dubos, 1950; Dubos and Mid­
dlebrook, 1948). Media based on a Tween
80-albumin formulation are satisfactory for
cultivation of tubercle bacilli for studies of
growth rates and kinetics and, in most
cases, for preparation of bacilli for chemi­
cal extraction. Furthermore, the basal me­
dia and supplements needed to prepare
such media as Dubos Tween-albumin broth
are readily available from commercial
sources.
ment represents an unwanted extrinsic pro­
tein that complicates the detection of the
small amounts of mycobacterial antigens
that are produced. Thorough washing of the
bacilli before their disruption and extrac­
tion can minimize this problem when in­
tracellular proteins are the substances of
interest. The problem associated with intro­
duction of the foreign albumin becomes
more serious when one is searching for
traces of extracellular proteins secreted by
the bacilli themselves into the medium.
Although most studies of diffuse growth
of M. tuberculosis in liquid medium have
relied on the presence of Tween 80 and
albumin, it is possible to obtain diffuse
growth with a detergent in the absence of
albumin. Wayne and Sramek used a dialy-
sate of the peptide-rich TB Broth Base
(Difco, Detroit, Mich.) as a base for a
Tween 80 medium that supported growth of
fairly large inocula of M. tuberculosis with­
out the addition of albumin (Wayne and
Sramek, 1979). Various Triton detergents,
which do not contain fatty acids, have been
employed to permit dispersed growth of
tubercle bacilli in the absence of albumin
(Dubos and Middlebrook, 1948); optimal

growth in the presence of Triton detergent
required the incorporation of the phospho­
lipid sphingomyelin in the medium.
Glycerol has commonly been used in
media when large crops of bacilli are de­
sired, since this carbohydrate markedly
stimulates growth of M. tuberculosis (note
that glycerol inhibits growth of fresh iso­
lates of the closely related M. bovis [Wayne
and Kubica, 1986]). However, there are
disadvantages to the use of glycerol. For
one, glycerol causes production of exces­
sive amounts of lipids and polysaccharides
in mycobacteria (Tepper, 1968), which may
interfere with isolation and purification of
other products of interest such as proteins
or nucleic acids (Hill et al., 1972). Addition­
ally, since it greatly stimulates oxygen con­
sumption by M. tuberculosis, the presence
of glycerol may cause such severe deple­
tion of dissolved oxygen as to lead to death
and autolysis of the bacilli in diffuse culture
when oxygen becomes limiting (Wayne and
Diaz, 1967).
Solid Media
A number of inspissated egg-based solid
media, including Lowenstein-Jensen, Wal-
lenstein, Petragnani, and variants thereof,
have been used widely for primary isolation
of tubercle bacilli from clinical specimens.
They tend to yield a higher proportion of
positives from direct clinical specimens
than do semisynthetic agar media (Kras-
now and Wayne, 1969). The egg media are
very rich and contain phospholipids and
proteins that tend to bind and/or neutralize
toxic products in clinical specimens. They
are not especially useful for research pur­
poses, being very complex and not repro­
ducible because of variation in the quality
of ingredients and the effects of heat in their
preparation.
Agar-based media
Agar media for growth of M. tuberculosis
are usually prepared from semisynthetic
Chapter 6 • Cultivation of At. tuberculosis 77
basal media enriched with supplements.
They offer better defined components than
egg-based media. Furthermore, they are
transparent, permitting early microscopic
detection of colonies and the observation of
morphologic detail. Agar media are used
mainly for the quantification of viable ba­
cilli in growth and survival studies and for
determination of drug susceptibility. Gen­
erally, growth of tubercle bacilli on agar
plates is stimulated by inclusion of a 5 to
10% C 0 supplement to the air in the incu­
2
bator.
Dubos oleic albumin agar is almost
equivalent to Dubos Tween 80-albumin me­
dium that has been solidified with agar
(Dubos and Middlebrook, 1947). The major
differences are that the Tween 80 is omitted
from the agar base and oleic acid is substi­
tuted for the glucose in the albumin com­
plex supplement. Middlebrook introduced
a number of small modifications (i.e., the
7H series) to Dubos oleic albumin agar to
enhance repair and accelerate detection of
damaged bacilli in clinical specimens (Rob­
erts et al., 1991). Both the original Dubos
and the modified Middlebrook types of me­
dia are available commercially, either al­
ready prepared or as dehydrated products
and sterile supplements.
Other media
Among the other "culture media" that
have been used for preparation of crops of
M. tuberculosis is the mouse lung. Bacilli
isolated in large amounts from the lungs of
infected mice have been compared bio­
chemically, physiologically, and immuno­
logically to bacilli isolated from conven­
tional culture, and striking differences have
been observed. It is beyond the scope of
this chapter to explore this subject further,
but the reader is referred to a comprehen­
sive review by one of the pioneers in the
field, William Segal (Segal, 1984).
78 Wayne
INOCULA
Once a strain or strains of M. tuberculo­
sis have been selected for study, a large
seed pool should be prepared, subdivided
into small aliquots, and maintained at
-70°C. That is, the culture should not be
maintained by serial passage from experi­
ment to experiment, as such passage would
cause it to undergo physiologic changes. A
fresh seed vial should be thawed and used
to inoculate a single-passage subculture to
provide a large inoculum for each experi­
ment, or it may be used directly without
subculture to inoculate the experimental
culture or to inoculate animals.
The seed pool can be prepared from a
dispersed, detergent-containing liquid me­
dium; from a young, thin veil of pellicle;
from an unagitated synthetic medium; or
from typical corded colonies from solid
medium. If the last two types of prepara­
tions are to be used, they should be well
suspended in medium containing a deter­
gent (e.g., 0.02% Tween 80) by use of a
tissue grinder or agitation in a sealed tube
with a few glass beads in a vortex-type
mixer (Wayne, 1962). For safety reasons,
an open tissue grinder is not recommended;
if it is necessary to use one, stringent pre­
cautions, including working in a biosafety
hood, must be taken to prevent escape of
infectious aerosols into the laboratory.
When the suspension is to be used to
inoculate experimental animals, especially
by the intravenous route, it is especially
important that the bacilli be very uniformly
dispersed, preferably as single cells with a
minimum of clumping. The deposition of
the bacilli in different organs of intrave­
nously inoculated mice is known to be
strongly affected by the degree of disper­
sion of the bacteria in the inoculum. Opti­
mal dispersion for this purpose may be
achieved by brief exposure of the suspen­
sion to a burst of moderate energy in a
sonicator. Care must be taken to avoid too
great an exposure, which may disrupt some
of the bacilli (Lefford, 1984).
TEMPERATURE
M. tuberculosis has a narrower range of
growth temperatures than most other my­
cobacteria (Wayne and Kubica, 1986). I
have studied the growth of different species
of mycobacteria in tubes of Dubos Tween-
albumin broth incubated in an aluminum
gradient temperature block (Wayne, 1970).
Incubation, with a single brief, daily shake,
of cultures of M. tuberculosis in this block
yielded little if any growth at temperatures
below 29°C (unpublished data) (Fig. 1). The
growth rate increased in a temperature-
dependent fashion over the 10° range up to
about a 39°C maximum and underwent a
precipitous decline at higher temperatures,
with no significant growth seen at 42°C. For
most practical purposes, M. tuberculosis
should be cultivated at 37°C to allow for
slight drifts in temperature without serious
impact on the growth.
AERATION AND AGITATION
For many years, when large crops of
tubercle bacilli were needed, they were
0,fi-i
25« 30' 35* <0* 45 s
TEMPERATURE (•C)
Figure 1. Eight-day growth of M. tuberculosis in
Tween-albumin broth incubated in a gradient temper­
ature block. Growth is expressed as optical absor-
bance (A ). Triplicate tubes w e r e incubated at each
5S0
temperature, and the results w e r e plotted as the mean
A,
sso with vertical bars spanning ± 1 standard devia­
tion.

grown as pellicles on the surfaces of un-
agitated shallow layers of one of the syn­
thetic media cited above. Alternatively,
they were stirred or aerated with a sparger,
resulting in distribution of large clumps of
growth throughout the medium. This latter
method is suitable for production of masses
of cells for extraction of a variety of the
chemical components of tubercle bacilli for
different purposes. It must be recognized,
however, that old, thickened pellicles and
large dispersed clumps do not represent
physiologically homogeneous populations
of cells. Oxygen and components of the
culture medium do not penetrate into the
center of the hydrophobic clumps, so these
cultures represent mixtures of cells in a
wide range of physiologic conditions from
rapidly replicating cells to dormant ones or
even dead, autolyzing cells. Nevertheless,
these preparations are good sources of
DNA, lipids, and polysaccharides. The pro­
teins, on the other hand, may be present in
various degrees of degradation, as they are
released from some bacilli that may be
autolyzing due to inadequate nutrition or
aeration (Turcotte, 1969a, b). This is prob­
ably true of RNA as well.
Far better control of aeration and the
physiologic state of tubercle bacilli in cul­
ture may be achieved through the use of
detergent-containing medium as described
above. Most published work in this area
has employed 0.02% Tween 80 as the de­
tergent and 0.5% bovine serum albumin
fraction V as a scavenger to protect the
bacilli from toxic effects of traces of oleate
released from the Tween 80.
Although M. tuberculosis obviously
grows most rapidly with maximum expo­
sure to air, as on the surface of an agar
plate, an anomalous inhibition of growth
has been observed consistently when cul­
tures in flasks of Tween 80-containing me­
dia are aerated on a rotary shaker-incubator
(Lyon et al., 1961). It has been proposed
that excess oleate production was the cause
of this phenomenon and that addition of
Chapter 6 • Cultivation of M. tuberculosis 79
glycerol reversed the effect by esterification
(Lyon et al., 1961). This seems an unlikely
explanation, since albumin is present in the
medium in an adequate amount to protect
against oleate. Although I also observed the
growth inhibition in flasks on a rotary
shaker, I have always been able to grow M.
tuberculosis in Tween-albumin medium in
culture tubes on a rotary shaker or in flasks
that were aerated by magnetic stirring. On
careful observation of the contents of flasks
agitated on a rotary shaker and on a mag­
netic mixer, I have come to the following
conclusions about the cause of the anoma­
lous failure of tubercle bacilli to grow on the
rotary shaker (unpublished observations).
Since the flasks are mounted in positions
around the center of rotation of these shak­
ers, centrifugal force creates a wave motion
of the medium, with a crest of medium,
followed by a contralateral trough, sweep­
ing around the perimeter of the flask. The
bacilli in the culture tend to accumulate at
the triphasic interface of air, glass, and
culture medium. As a crest of medium
sweeps past a given point on the glass wall,
some of the cells are deposited on the glass
and marooned there as the crest passes.
The next pass of the crest deposits more
cells, which physically push the previous
deposit up the flask wall until, on succes­
sive revolutions, most of the bacilli in the
medium become trapped in a veil of cells on
the flask wall out of reach of fresh medium.
The apparent protective role of glycerol
may thus be an effect on surface tension of
the medium. Behling and colleagues (Beh-
ling et al., 1993) have recently demon­
strated that when trehalose 6,6'-dimycolate
(cord factor) extracted from M. tuberculo­
sis is coated onto inert particles and sus­
pended in saline in a test tube, the particles
aggregate in veils that climb the wall of the
container. This cord factor effect suggests a
mechanism for the peculiar tendency of
tubercle bacilli to accumulate at the tripha­
sic interface and climb the walls of shaken
flasks.
80 Wayne
In contrast to the failure of flasks of
medium on a rotary shaker to support
growth of M. tuberculosis, luxuriant
growth occurs when a magnetic stirrer is
used to agitate the contents of a flask. The
crest-and-trough wave effect is absent in
the magnetically stirred medium; instead, a
vortex forms and continuously transports
the tubercle bacilli back into the aerated
medium, where they are bathed in nutrients
and grow rapidly. In test tubes on a rotary
shaker, the surface-to-volume ratio seems
to be so low that the wave effect is incon­
sequential, and the bacilli remain sus­
pended in the medium and grow rapidly.
Similarly good growth is obtained in roller
bottle cultures in Tween-containing 7H9
medium.
VIABILITY
The viability of mycobacterial cultures
varies greatly with conditions. In the case
of BCG vaccines, the total number of or­
ganisms present in a culture is generally
determined by microscopic counting in spe­
cial bacterial counting chambers, e.g.,
Petroff-Hauser chambers, and the number
of viable organisms is determined by count­
ing the CFU on solid agar medium. As
described in chapter 31, BCG vaccines pre­
pared as pellicle cultures have viabilities
generally between 5 and 20% (Mackaness
et al., 1973; Gheorghiu et al., 1988). Bacte­
ria grown under optimal conditions (for
example, in roller bottles) can yield cul­
tures in which virtually every physical ba-
cillary particle is viable and is a CFU (Mac­
kaness et al., 1973). Mycobacteria survive
freezing at -70°C with little loss in viabil­
ity, but they lose 50% or more viable CFU
by freeze-drying.
MEASUREMENT OF GROWTH AND
GROWTH DYNAMICS
Measurement of Growth
When tubercle bacilli are grown in deter­
gent-free medium, it is very difficult to
measure rates of growth, since different-
sized clumps contain bacilli in different
physiologic states. Optical or colony-count­
ing methods are essentially useless with
hydrophobic clumps. The major approach
has been filtration or centrifugation and
measurement of wet or dry weight of the
crop (Bowles and Segal, 1965).
When accurate measures of growth dy­
namics are needed, it is far better to use
diffuse cultures in a detergent-containing
medium. The simplest method of measuring
growth rates is determination of the optical
absorbance of cultures in tubes or sidearm
flasks, since these flasks do not require the
opening of the culture at each reading and
therefore diminish the risk of contamina­
tion. It has been my practice to distribute
Dubos Tween-albumin broth in 10-ml
amounts to screw-cap culture tubes (20 by
125 mm) for agitated or stationary culture
(see below). The growth is measured by
determining t h e ^ l in a Coleman Jr. spec­
580
trophotometer (Coleman Instruments,
Maywood, 111.). In this system, an A of
580
0.100 corresponds to a count of 6.3 x 10 7
CFU of M. tuberculosis H37R, per ml
(Wayne, 1976). The A of a culture is a
580
linear function of cell concentration over a
range of readings up to y 4 = 0.200. At
580
higher cell concentrations, particle coinci­
dence effects cause a departure from linear­
ity. However, it is not difficult to prepare a
correction curve by using dilutions of a
barium sulfate suspension of known con­
centrations, which allows adjustment of ob­
served to true A readings. This permits
5S0
plotting of growth curves of M. tuberculo­
sis of remarkable reproducibility and excel­
lent correlation with plate counts over a
wide range of growth (Wayne, 1976).
While optical measurements are usually
sufficient for following growth dynamics
once a correlation has been established
with actual counts of CFU per milliliter,
studies of survival or of very early growth
of tubercle bacilli can be done accurately
only by plating dilutions of liquid cultures

or suspensions to solid medium and deter­
mining CFU per milliliter by direct colony
counts. If the original culture material has
been prepared in detergent-containing me­
dium, serial 10-fold dilutions may be made
directly from the culture into detergent-
containing diluent. One can use the com­
plete Dubos Tween-albumin broth as a di­
luent, or if the dilutions are made and plated
rapidly, it is more economical to use the
Dubos broth base with Tween 80 but with­
out albumin as diluent. If the original cul­
ture material is not in a well-dispersed
form, it is necessary to grind it gently in the
detergent-containing media by vortex-type
mixing in a sealed tube with glass beads
(Wayne, 1962) or in a tube-and-pestle type
of tissue grinder; in the latter case, strin­
gent precautions must be taken to prevent
escape of infectious aerosols into the air of
the laboratory. Optimal dispersion of tuber­
cle bacilli may be achieved by brief, mod­
erate-energy sonication of the suspension
(Lefford, 1984). The upper end of the range
of dilutions needed for a given experiment
may be estimated from the turbidity of the
original suspension on the basis of a previ­
ously established constant correlating ^ 4 580
in the 0.10 to 0.20 range with dilution plate
counts.
After the dilutions have been made, they
should be inoculated to one of the conven­
tional solid media, such as Dubos oleic-
albumin agar or one of its derivative media.
The medium is dispensed in 5-ml amounts
to each of the segments of a quadrant petri
dish or in 3-ml amounts to the wells of a
24-well tissue culture plate and allowed to
gel. If petri plates are used, each quadrant
should receive 50 u.1 of one of the dilutions
of inoculum dotted about the surface by a
calibrated micropipettor fitted with sterile
disposable tips; if tissue culture plates are
used, the inoculum should not exceed 20 u,l
per well. The agar should be incubated at
37°C in a well-humidified chamber, and
colonies should be counted at 10 x to 100 x
power twice a week until counts have sta­
Chapter 6 • Cultivation of M. tuberculosis 81
bilized. Colonies of M. tuberculosis first
appear as dense dots but soon produce
corded fringes that are characteristic of the
species. The original concentration of via­
ble bacilli is calculated from the size of the
inoculum plated and the number of colonies
seen in those dilutions that yield a final
count of 10 to 100 colonies per quadrant or
10 to 50 colonies per well.
Growth Dynamics
The decision as to whether to use stirred
or unagitated cultures should be made ac­
cording to the purposes to which the cul­
ture is to be put and an understanding of the
different physiologic phases that M. tuber­
culosis manifests under various conditions
of aeration.
When M. tuberculosis is grown dispersed
in continuously agitated liquid medium, it
exhibits the expected logarithmic growth,
with doubling times of 16 to 18 h (Wayne,
1976). Unexpectedly, when comparable cul­
tures are not agitated continuously but are
simply shaken briefly to permit turbidimet-
ric determination of growth, the growth ap­
pears to conform to a simple arithmetic mode
(Fisher and Kirchheimer, 1952) rather than
merely shifting to a slower rate of geomet­
ric growth; this phenomenon is difficult to
understand when dealing with organisms
that replicate by binary fission. Volk and
Myrvik (1953) suggested that this could be
explained by a slowing down of the replica­
tion of tubercle bacilli in oxygen-limiting
unstirred culture until the rate of settling of
bacilli equaled the rate of replication, with
the bacilli that had settled to the bottom of
the tube having ceased replicating entirely.
My colleagues and I subsequently con­
firmed this explanation and established a
number of physiologic events that charac­
terize this phenomenon (Wayne, 1976,
1977; Wayne and Lin, 1982; Wayne and
Sramek, 1979). When small inocula of tu­
bercle bacilli were dispersed in tubed media
and incubated without any agitation, the
82 Wayne
initial growth occurred at a logarithmic
rate, with doubling every 16 to 18 h until the
cell density approached 4 x 10 CFU/ml.
7
Thereafter, dissolved oxygen became limit­
ing. By the use of inocula stably labeled
with C , it was established that replication
1 4
in the unstirred culture slowed down until it
reached a doubling time of 33 h, at which
time the logarithmic doubling in the upper
layers was exactly balanced by the rate at
which bacilli settled to the bottom of the
tube (Wayne, 1976). Thus, there was a
constant population of slowly dividing cells
in suspension and an arithmetically increas­
ing mass of bacilli in the sediment. Further­
more, the bacilli that accumulated in the
sediment had undergone a discrete meta­
bolic shiftdown during their transition
through the self-generated oxygen gradient
in the tube and were in a uniform state of
dormancy. When the bacilli in the sediment
were resuspended and diluted into fresh
aerated medium, they immediately initiated
RNA synthesis. Between hours 8 and 12
after reconstitution, the bacilli underwent a
cycle of synchronized division, and only
then did they initiate DNA synthesis
(Wayne, 1977). Synchronized replication
was sustained through at least three 20-h
cycles. The dormant bacilli that accumu­
lated in unstirred cultures were far more
resistant to the bactericidal effects of anaer-
obiosis than were actively aerated cultures
after being abruptly deprived of oxygen
(Wayne and Lin, 1982). As tubercle bacilli
in unstirred cultures shifted down while
settling through their self-generated oxygen
gradient, they accumulated an antigen that
was not found in aerated cultures (Wayne
and Sramek, 1979) and exhibited a 4-fold
increase in isocitrate lyase and later a 10-
fold increase in a glycine dehydrogenase
that may have been instrumental in scav­
enging sufficient NAD for completion of the
final round of DNA synthesis before dor­
mancy was established (Wayne and Lin,
1982).
Inasmuch as tubercle bacilli in the mam­
malian host occur in intracellular environ­
ments and in conditions of inflammation
and necrosis, the highly aerobic in vitro
conditions of shaken cultures or the sur­
faces of solid medium do not reflect the
state of the bacilli as they occur in the
infected host. M. tuberculosis is known to
produce enzymes of anaerobic metabolism,
and these are especially prominent when
bacilli are tested after direct harvest from
host tissues; there is a general shift away
from 0 -dependent pathways to anaerobic
2
or facultative anaerobic pathways in host-
derived bacilli compared to cultured organ­
isms (Segal, 1984). The availability of the in
vitro model of shiftdown to dormancy and
of synchronized shiftup from dormancy to
rapid replication of M. tuberculosis through
manipulation of the degree of aeration of
cultures, as described above, should make
it possible to study the physiology, immu­
nology, and chemistry of these bacilli in
phases that reflect their condition in various
stages of activity and dormancy (latency) in
human disease.
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