Identification
of Mycobacterial
Colony
Pathogens
Utilizing
Characteristics
ERNEST H. RUNYON, P H . D .
Microbiology Research, Veterans Administration Hospital, Salt Lake City, Utah S4113
ABSTRACT
Runyon, Ernest H.: Identification of mycobacterial pathogens utilizing colony
characteristics. Amer. J. Clin. Path. 54: 578-586, 1970. Morphologic properties
of colonies growing on either 7H10 or corn meal glycerol agar plates, or
both, are of assistance in identification of mycobacterial pathogens. By examination of microscopic colonies, early reporting of Mycobacterium tuberculosis
may be made, and common difficulties from contaminated cultures can be
avoided. Colonies of M. kansasii have a distinctive SR morphology and exhibit
crystal production as well as photochromogenicity. Colonies of Battey bacilli
are usually sharply distinct, being thin, smooth, and translucent. M. xenopi is
recognized by slowly developing yellow colonies which, by microscopic examination, can be seen to be surrounded with projecting filaments. M. abscessus can most easily be distinguished from the other common rapid grower,
M. fortuitum, by the absence of branching filamentous extensions from its
colonies.
T H E DISTINCTIVE APPEARANCES of colonies
of various species of mycobacteria have
been shown in published figures a number
of times, 3-9 ' 12>14 but microscopic examination of colonies appears to be infrequent
in clinical laboratories. M. tuberculosis, M.
kansasii, M. intracellular is, M. xenopi, M.
abscessus, and M. fortuitum can be distinguished from each other simply by examining the colonies under low power. Biochemical tests are ancillary. Colonies are
grown on suitable transparent plated media
and examined inverted on the stage of a
low-power microscope. Such plate cultures
permit the earliest possible reporting of
M. tuberculosis (Figs. 1 and 2). They permit definite recognition of the presence or
absence of contaminants (Fig. 3). Often
these are not evident on egg medium
slants and lead to errors in testing for niaReceived December 15, 1969; accepted for publication February 12, 1970.
cin and drug susceptibility. Plates also
provide a means of obtaining pure cultures
by single-colony selection. Early and unequivocal evidence of mycobacteria other
than tubercle bacilli is provided if these
are present. Their colonies usually are
sharply different from those of tubercle
bacilli. Colonies of Battey bacilli, for example, are regularly transparent, thin, and
smooth. Battey bacilli have often been reported as tubercle bacilli, but no microbiologist will make this mistake if he looks
at plate-grown colonies. Carotene crystals
and branching filamentous extensions, or
the lack of these, provide means of recognition of other species.
Planting, Incubating, and Examining
Plate Cultures
Materials
Plastic petri dishes. Glass dishes are satisfactory only if bottoms are plane and of
uniform thickness.
578
October 1970
579
MYCOBACTERIAL COLONY CHARACTERISTICS
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Fie. 1 (left). Tightly corded texture o£ Mycobacterium tuberculosis. Age three weeks, 7H10.
X 120.
FIG. 2 (center). Ten-day-old colonies (subculture) of M. tuberculosis on 7H10. X 120. Curving
strands on young colonies as seen here are not alone definitive of M. tuberculosis.
Fie. 3 (right). Two colonies of a contaminant. X 120. Contaminants, often obscure on egg
medium, make drug susceptibility and biochemical tests unreliable. Plates provide prominent
evidence of contaminants if present, and easy means of isolating pure cultures.
Translucent agar media, like 7H10 of
Middlebrook and Cohn, and corn meal
(CM) agar with 3 % glycerol.
Plastic bags or other containers to conserve moisture and C 0 2 if this is added.
Low power microscope.
Method
Part of the specimen digest (e.g., sputum
concentrate) may be planted directly on
plates of 7H10 medium as well as on tubed
or bottled egg medium, such as Lowenstein-Jensen. After incubation for 5 to 14
days, at which time no colonies may be
grossly visible, the plates are inverted on
the microscope stage and examined, using
objectives such as 3.5 X and 10 X. It will
often be immediately apparent whether
microscopic colonies are typical of tubercle
bacilli or not. If not, each colony of bacilli
of the acid-fast type is immediately transferred to CM and to other 7H10 plates by
streaking, to attain isolated colonies. The
plates are incubated for 2 days to several
weeks, depending on amount and charac-
ter of growth obtained. Colonies which
grow rapidly should be examined at 24
hr.; usually they will be mature in 2 to 3
days. M. ulcerans requires more than 4
weeks; most other species require 1 to 3
weeks.
Colony
Interpretation
Stage of development of colonies is important. Transient early stages may be distinctive. Full maturity may be attained
only where colonies are well isolated, but
diagnostically important stages may occur
also in more closely seeded areas where
colonies sometimes remain immature.
Roughness of colony is manifested by
cohesion of bacilli, usually in curving
strands. These diffract transmitted light.
Thus, roughness is one condition which
causes colonies to appear (with a low
power microscope) dark rather than translucent. No code for colony description is
used here except the universally accepted
designations "R" and "S" for "rough" and
"smooth."
580
RUNYON
Rhizodes (taken from the word rhizoids,
homology with which is not implied) is a
term we have used for the portion of colonies which penetrates the medium. Dark
centers of colonies are usually rhizodes, as
can be demonstrated by pushing away the
other colony parts with a needle or swab.
Rhizodes of mycobacteria may be present
or absent, depending upon age, species,
and medium; their shape may be peg-like,
bulbous, or of many other forms, including
some very similar to the substrate mycelium of Nocardia.
Precautions
Avoid liquid in plates or in plate containers. The formation of more than a thin
film of condensate is not to be tolerated. If
visible liquid is present in bags or other
plate containers, these should be autoclaved unopened and discarded, or opened
with due care in a transfer hood. Avoidance of liquid is not difficult but requires
vigilance. Liquid inocula should be small
in volume, and a period allowed for absorption into the medium before inversion
of plates. Condensation is avoided by keeping the plates always in a position such
that the lids are, if anything, a little
warmer than the contained medium. Bags
of plates should be opened promptly upon
removal from the incubator. At the microscope an electric heating pad is of great
assistance in providing gentle ascending
heat for stacked inverted culture dishes.
Although usually unnecessary, it may be
desirable to place each culture plate in an
individual polyethylene bag; examination
for growth may then be made without removal of the plate from the bag.
Transferring from plates containing tubercle bacilli should, of course, be done
under a transfer hood, as should any transfer of tuberculous material. Hoods with
provision for stereomicroscopic examination of plates, with only the oculars of the
microscope projecting are available. Such
A.J.C.P.—Vol. 54
hoods facilitate transfer from single colonies.
For best growth of M. tuberculosis on
7H10 medium, an increase in the C 0 2 concentration to 5 to 10% is recommended.
Note that plastic bags which are permeable
to C 0 2 (polyethylene) or C0 2 -impermeable
(e.g., Mylar) may be used, depending upon
whether C 0 2 is put into the bags only or
into the whole incubator atmosphere.
Properties for Identification of
Mycobacterial Pathogens
M. tuberculosis
Colonies of tubercle bacilli invariably
have a prominent patterned texture, due
to tight cording of the bacilli (Figs. 1 and
2). Typical growth may often be recognized in less than 2 weeks, before any
grossly visible colonies appear, so an early
report, "Colonies resembling M. tuberculosis" may be made, with note that a report of definite species identification will
follow.
Other properties required to be known
for definitive identification are slow
growth, a positive niacin test,11 and at
least one of the following: (1) nitrate reduction, 12 (2) negative catalase after heating at 68 C.,10 and (3) typical drug susceptibility.
Other species that have similar colonies
are rare in clinical material but should be
anticipated. Some indication of a species
other than M. tuberculosis may be the
presence of some smooth colonies or incompletely rough ones. If bacilli from such
colonies are acid-fast or partially so, they
should be streaked on CM and on other
7H10 plates, and other tests made. An encounter with a rough colony, niacin-positive strain of other species should be recognized as a possibility. Niacin-positive
rough colony Group IV strains are well
known (M. borstelense var. niacinogenes).
Such strains are distinct from tubercle bacilli in most other properties, as persistence
October 1970
MYCOBACTERIAL COLONY CHARACTERISTICS
of catalase after heating at 68 C , 3-day
arylsulfatase activity, drug resistance, ability to grow on CM, and at 25 C.
M. bovis
M. bovis in the United States occasionally occurs in specimens of veterinary origin, but rarely in human sources. It grows
poorly, if at all, on media lacking pyruvate
and containing more than 0.5% glycerol.
Colonies of M. bovis resemble M. tuberculosis in tight cording, but remain thin. M.
bovis is niacin-negative by the usual test
and is uniquely susceptible to thiophene-2carboxylic acid hydrazide. 1
M. kansasii
M. kansasii is universally recognized by
yellow pigmentation, which develops only
if the growing cultures are exposed to
light. If plates are incubated in continuous
light, M. kansasii forms crystals of carotene,
and the appearance of these on or in colonies from a specimen source other than a
superficial body area (as sputum) is alone
enough for almost certain identification of
M. kansasii (Fig. 5).
Colony centers are thickened (darker);
the thinner peripheral portions show more
or less stranding of bacilli. Strains vary in
roughness, commonly are intermediate (SR
. . . RS), but may be completely rough or,
rarely, fully smooth. As seen by reflected
light (stereomicroscope) the colonies have
a characteristic sheen produced by a discontinuous surface (waxy?) film. This distinctive feature is present irrespective of
whether the M. kansasii colonies are pigmented. Growth on CM is very poor or
lacking (Figs. 4 to 6).
Occurrence of nonphotochromogenic M.
kansasii is extremely rare. Scotochromogenic strains are distinctive in producing
exceedingly abundant crystals of carotene,
an appearance never seen on the usual
Group II strains. Nonpigmented M. kansasii strains have the typical colony mor-
581
phology of the species and, in contradistinction to M. gastri (which has somewhat
similar colonies), reduce nitrate and have
active catalase after heating at 68 C.
M. marinum
Colonies are similar to those of M. kansasii, but usually they are smoother and
rhizodes are more readily seen (Fig. 7). Distinguishing features: (1) source never in
sputum; always from a superficial lesion;
(2) at 37 C. poor or no growth on initial
isolation; at 25 C. growth is much more
rapid than that of M. kansasii; (3) nitrate
not reduced.
M. ulcerans
M. ulcerans is recognized by its source
(superficial lesion on person from tropical
area), growth restriction to 32 to 33 C ,
and by very slow growth (often 2 months
or more). Niacin test may be positive; colonies show serpentine stranding.
M. scrofulaceum complex (Group II)
Colonies are scotochromogenic, pigmented when first discernible. No colony
characteristic which reliably distinguishes
scotochromogenic strains which are at times
pathogenic, from those which only very
rarely are, has been identified. The former
usually fail to hydrolyze Tween. If colonies are very thin and flat, suspicion of
possible pathogenicity may be greater than
if colonies are erect and domed. This,
however, is conjecture, based on possible
parallelism with M. intracellular is.
M. intracellulars complex (Group III)
Colonies, in sharpest contrast to M. tuberculosis, are usually thin, nearly or completely transparent, in early growth structureless, sometimes radially lobed ("asteroid"), especially on CM; on 7H10 usually
nearly or quite entire (without indentations), circular in outline (Figs. 8 and 9).
Colonies of the Battey complex only 3 to 6
582
RUNYON
A.J.C.P.— Vol. 54
FIG. 4 (upper, left). Serpentine stranding (young colony), M. kansasii. X 120.
FIG. 5 (upper, right). A light-grown colony of M. kansasii showing flakey surface crystals
(arrow) and granular submerged crystals (dark dots). X 120.
FIG. 6 (lower, left). Rough colony of M. kansasii. X 120.
FIC. 7 (lower, right). M. marinum showing loose cording. Rhizode in center of colony, x 200.
October 1910
MYCOBACTERIAL COLONY CHARACTERISTICS
583
Fie. 8 (upper, left). On
CM, colonies o£ M. intracellularis complex are
thin and transparent (unless rough). Age 2 to 3
weeks; note rhizodes. X
90.
Fie. 9 (upper, right). On
7H10, colonies o£ M. intracellularis are usually
entire and circular, with
or without a thin peripheral skirt. X 90.
FIG. 10 (lower, left). An
occasional rough colony
of M. intracellularis may
appear, x 90.
FIG. 11 (lower, right).
Thicker, domed colonies
of M. intracellularis in
subcultures are mutants.
X90.
days old may show a texture resulting
from a transient elongation of component
bacilli.
The most common human pathogen
having colonies of this type in America
and in some other countries is M. intracellularis (Battey bacillus). M. avium also
occurs as a human parasite, but very rarely
so in the United States. Except for epidemiologic investigations, it is usually unnecessary to determine if a given strain is
M. avium or M. intracellularis. Colonies of
saprophytes of Group III (M. terrae, M.
gastri, and others) 16 are often granular or
otherwise different from those described,
but it must be recognized that Battey ba-
584
RUNYON
A.J.C.P.—Vol. 5-1
yellow. Cells include many which are very
long and thin. No growth occurs at 25 C ,
nor in media containing isoniazid or streptomycin, 5 fig. per ml. (Fig. 13).
FIG. 12. Colonies of M. avium often show rough
or RS variants. X 90.
cilli as well as saprophytes which appear
similar may occur in sputum as contaminants, not related to disease.
The most definitive supplementary tests
are immunologic, for example, agglutination serogrouping. 2 - 13 Tween hydrolysis
should be determined: only nonpathogens
are positive; Battey and avian bacilli are
negative.
A few rough (dark) colonies may appear,
together with the much more prevalent
smooth ones (Figs. 10 to 12). Rarely, all of
a Battey patient's colonies will be rough.
Such strains might not be typable by agglutination. They are distinct from M. tuberculosis in being niacin-negative, nitratereduction-negative, catalase-positive at 68 C ,
and drug-resistant; they are distinguished
from nonpathogens by being Tween-hydrolysis negative. Furthermore, the colonies, while being similar to those of tubercle
bacilli, on careful examination may be
seen to have branching filaments. These
appear in early growth and at times extend from margins of confluent mature
growth. Such filamentous growth does not
occur with M. tuberculosis.
M. xenopi
Colonies of slow growth that regularly
have a persistent fringe of filaments on CM
are a very distinctive feature of this species.
Colonies are small and, at maturity, usually
FIG. 18 (upper). Mature colony of M. xenopi,
age 4 weeks, on CM. Note projections from the
colonies. X 90.
FIG. 14 (middle). No filamentous extensions of
colonies of M. abscessus on either CM or 7H10.
Rough colonies. X 100.
FIG. 15 (lower). Same as Figure 14. Smooth colonies, x 100.
October 1970
MYCOBACTERIAL COLONY CHARACTERISTICS
585
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1
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t
FIG. 16 (upper, left). Colonies of M. fortuitum filamentous on either CM or 7H10, 1 day
old. X 100.
FIG. 17 (upper, right). Older colonies of M. fortuitum on 7H10 may show no filaments. Dark
centers are rhizodes. X 160.
FIG. 18 (lower, left). Young mycelial colony of M. fortuitum showing branching filamentous
rhizodes (dark lines), CM. X 160.
FIG. 19 (lower, right). Eight-day-old colony of M. fortuitum on CM, with persistent branchingfilamentousextensions. X 150.
Rarely, M. xenopi lacks yellow color. If M. fortuitum, which on primary culture
nonpigmented colonies with branching fil- may not exhibit the characteristic rapid
amentous extensions appear, these may be growth. Other tests mentioned above must
586
RUNYON
be made. M. fortuitum grows readily at
25 C. and is resistant to 5 /ig. per ml.
isoniazid.
M. fortuitum,
M. abscessus (Group IV)
Occasionally a strain of one of the species of rapidly-growing bacteria does not
grow rapidly on primary isolation. Other
properties such as colonial appearance may
help in identification; mutation in subcultures will soon result in the characteristic rapid growth. Species delineation in
this area is far from settled. Two species
or species-complexes are recognized among
the arylsulfatase-positive, potential pathogens: M. fortuitum and M. abscessus. M.
fortuitum regularly produces mycelial colonies on either 7H10 or CM in 24 hr. M.
abscessus does not. Fully mature colonies
are seen after 3 or 4 days. On CM many
colonies of M. fortuitum will be found still
to have branching filamentous extensions,
some of these on the agar surface, others
penerating into the medium as a kind of
substrate mycelium. On 7H10 or other rich
media, mature colonies ordinarily do not
show filamentous extensions. A few other
species of rapid growers have similar colonies (e.g., M. smegmatis), but these species
almost never appear in clinical specimens,
and are distinct in being arylsulfatasenegative (Figs. 14 to 19).
M. abscessus, whether of smooth or of
rough colony type, is distinctive in lacking
branching filamentous extensions. The colonies are thin, with a fine granular texture.
On 7H10 the colonies are circular in outline, flat at first, then become low domed.
On CM smooth colonies usually are radially lobed. Rhizodes, conspicuous on M.
fortuitum, are absent or inconspicuous on
M. abscessus.
A.j.C.P.—Vol. 5-1
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