Mycobacteria CHAPTER 25

▪ Mycobacterium microti
 Mycobacterium leprae (Hansen’s bacillus)
 Atypical mycobacteria or nontuberculous mycobacteria (NTM)
or mycobacteria other than tubercle bacillus (MOTT)

RUNYON’S CLASSIFICATION OF MOTT

 Group I (Photochromogens)

▪ Mycobacterium kansasii – also known as yellow

bacilli or shepherd’s crook
▪ Mycobacterium marinum – causes swimming pool
granuloma and are usually found in sea water
▪ Mycobacterium simiae – causes pulmonary/TB-like
GENERAL CHARACTERISTICS infection in monkeys
▪ Mycobacterium asiaticum
 Mycobacteria are slender, slightly curved or straight, rod-
shaped organisms 0.2 to 0.6 μm × 1 to 10 μm in size  Group II (Scotochromogens)
 Non-motile and do not form spores
 Cell wall has extremely high lipid content; thus, mycobacterial ▪ Mycobacterium scrofulaceum – causes cervical
cells resist staining with commonly used basic aniline dyes, such lymphadenitis, called scrofula
as those used in Gram stain, at RT ▪ Mycobacterium szulgai
 Mycobacteria take up dye with increased staining time or ▪ Mycobacterium gordonae – also called tap-water
application of heat but resist decolorization with acid-ethanol – bacillus
a characteristic referred to as acid fastness – hence, the term ▪ Mycobacterium flavescens
AFB
 Acid fastness is a basic characteristic in distinguishing  Group III (Non-photochromogens)
mycobacteria from most other genera
 Strictly aerobic, but increased CO2 will enhance the growth of ▪ Mycobacterium avium complex – also known as
some species Mycobacterium intracellulare or Batley’s bacillus
 Pathogenic mycobacteria grow more slowly than most other ▪ Mycobacterium gastri – also called J bacillus
bacteria pathogenic for humans ▪ Mycobacterium terrae complex – also called radish
 Rapidly growing mycobacteria generally grow on simple media bacillus
in 2 to 3 days at temperatures of 20°C to 40°C ▪ Mycobacterium terrae
 Most mycobacteria associated with disease require 2 to 6 ▪ Mycobacterium triviale
weeks of incubation on complex media at specific optimal ▪ Mycobacterium nonchromogenicum
temperatures ▪ Mycobacterium xenopi
 One of the mycobacteria pathogenic for humans, M. leprae, ✓ Colonies appear like bird’s nest with stick-like
fails to grow in vitro projections in cornmeal agar
✓ Scotochromogenic and thermophilic
MAJOR GROUPS OF MYCOBACTERIA ▪ Mycobacterium celatum
▪ Mycobacterium genavense
 Mycobacterium tuberculosis complex ▪ Mycobacterium haemophilum
▪ Mycobacterium tuberculosis (tubercle bacilli) ▪ Mycobacterium malmoense
▪ Mycobacterium bovis (including the vaccination strain ▪ Mycobacterium ulcerans
bacillus of Calmette-Guérin) ✓ Third most common mycobacterium after
▪ Mycobacterium africanum Mycobacterium tuberculosis and Mycobacterium
▪ Mycobacterium canettii leprae

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Mycobacteria CHAPTER 25

✓ Causes Buruli ulcer in Africa and Bairnsdale ulcer  One of the mycobacteria pathogenic for humans, M.
in Australia genavense, does not grow on media used routinely to isolate
 Group IV (Rapid Growers) mycobacteria and requires extended incubation (6 to 8 weeks)
▪ Non-pathogenic group which grows in less than 7 days  M. leprae and M. microti fail to grow on artificial media
▪ Mycobacterium fortuitum – non-photochromogens  Many different media available for the recovery of
▪ Mycobacterium chelonae – non-photochromogens; mycobacteria from a clinical specimen are variations of three
formerly named general types:
▪ Mycobacterium borstolense ▪ Egg-based media
▪ Mycobacterium smegmatis ▪ Serum albumin agar media
▪ Mycobacterium phlei – scotochromogen; also called Hay ▪ Liquid media
bacillus  Use of a solid-based medium, such as LJ medium, in
▪ Mycobacterium vaccae combination with a liquid-based medium is recommended for
routine culturing of specimens for the recovery of AFB

STAINING FOR ACID-FAST BACILLI

 When Gram-stained, Mycobacterium spp. stain faintly or not at

all, giving a beaded appearance because of irregular uptake of
the stain caused by the increased lipid content of the cell wall
 Acid-fast smears are prepared directly from clinical specimens
and from digested, decontaminated, and concentrated
specimens
 Ziehl-Neelsen (hot method)
 Kinyoun (cold method)
 Auramine-rhodamine (bright yellow color) – fluorescent stain
 FITC (fluorescein isothiocyanate) – fluorescent stain
 When examining acid fast-stained smears, examine at least 300
fields for 15 minutes
 Other acid-fast bacteria include:
▪ Legionella micdadei
▪ Rhodococcus
▪ Nocardia
EGG-BASED MEDIA
CULTURE MEDIA AND ISOLATION METHODS  Basic ingredients in an inspissated egg medium are fresh whole
eggs, potato flour, and glycerol, with slight variations in defined
salts, milk, and potato flour
 Mycobacteria are strictly aerobic and grow more slowly than  Each contains malachite green to suppress the growth of gram-
most bacteria pathogenic for humans positive bacteria
 M. tuberculosis has the longest replication time, at 20 to 22  Glycerol enhances growth of Mycobacterium avium complex
hours  Löwenstein-Jensen (LJ) medium is the medium most commonly
 The rapidly growing species generally form colonies in 2 to 3 used in clinical laboratories and the medium commonly used
days, whereas most pathogenic mycobacteria require 2 to 6 for niacin test
weeks of incubation  Examples are Löwenstein-Jensen, American Trudeau Society
 Growth of M. tuberculosis is enhanced by an atmosphere of 5% (ATS) medium, Petragnani, Dorset egg medium, Dubois Tween
to 10% CO2 Albumin, and Wallenstein’s medium
 Mycobacteria require a pH between 6.5 and 6.8 for the growth
medium and grow better at higher humidity

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Mycobacteria CHAPTER 25

AGAR-BASED MEDIA ▪ Plate should be incubated at 30°C – optimal temperature

for recovery of this organism and for M. marinum
 Serum albumin agar media, such as Middlebrook 7H10 and
 Alternatively, hemin (X) strips or disks used for the
7H11 agars, are prepared from a basal medium of defined salts,
identification of Haemophilus spp. can be placed on
vitamins, cofactors, glycerol, malachite green, and agar
Middlebrook agar plates
combined with an enrichment consisting of oleic acid, bovine
 Septi-Chek AFB
albumin, glucose, and beef catalase (Middlebrook OADC
▪ Biphasic media system for the detection and isolation of
enrichment)
mycobacteria
 Middlebrook 7H11 medium also contains 0.1% casein
▪ System consists of a bottle containing Middlebrook 7H9
hydrolysate, which improves the recovery of isoniazid-resistant
broth with an atmosphere of 5% to 8% CO2, an enrichment
strains of M. tuberculosis
consisting of growth-enhancing factors and antimicrobial
 The addition of antimicrobial agents to 7H10 or 7H11 makes
agents, and a paddle with agar media
the media more selective by suppressing the growth of
▪ One side of the paddle is covered with nonselective
contaminating bacteria
Middlebrook 7H11 agar
 Mitchison’s selective 7H11 contains polymyxin B, amphotericin
▪ One of the two sections on the reverse side of the paddle
B, carbenicillin, and trimethoprim lactate
contains a modified egg-based medium for differentiating
 Advantages of clear agar-based media over opaque egg-based
M. tuberculosis from other mycobacteria
media:
▪ The other side contains CHOC agar for the detection of
▪ Can be examined using a dissecting microscope for early
contaminating bacteria
detection of growth and colony morphology
▪ Biphasic media system provides for rapid growth and
▪ Drug susceptibility tests may be performed on agar-based
identification and drug susceptibility testing, without the
media without altering drug concentrations, which occurs
need for routine subculturing
with egg-based media
▪ Biphasic media system is sensitive and does not require
▪ When specimens are inoculated to Middlebrook 7H10 and
the use of radioactivity and a CO2 incubator
7H11 media and incubated in an atmosphere of 10% CO2
▪ Detection times are shorter than with conventional agar
and 90% air, 99% of the positive cultures are detected in 3
but significantly longer than with the BACTEC system
to 4 weeks, earlier than for those detected on egg-based
media

LIQUID-BASED MEDIA

 Mycobacterium spp. grow more rapidly in liquid medium, and it

can be used for both primary isolation and subculturing
 Middlebrook 7H9 broth and Dubos Tween albumin are
nonselective liquid media used for:
▪ Subculturing stock strains
▪ Picking single colonies
▪ Preparing inoculum for in vitro testing
 Middlebrook 7H12 and Middlebrook 7H13

OTHER CULTURE MEDIA FOR RECOVERY OF ISOLATOR LYSIS-CENTRIFUGATION SYSTEM

MYCOBACTERIA
 CHOC agar plate
▪ Should be included in the primary isolation media for skin
and other body surface specimens for the recovery of M.
haemophilum, which requires ferric ammonium citrate or
hemin for growth
 Isolator is a blood collection system that contains saponin to
liberate intracellular organisms
 After treatment with the saponin, the sample is inoculated onto
mycobacteria media plates or tubes
 Advantages:
▪ System allows for higher yields and shorter recovery times
for mycobacteria than conventional blood culture methods

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Mycobacteria CHAPTER 25

▪ Yield isolated colonies TEMPERATURE

▪ Ability to quantify mycobacteremia – useful in monitoring
effectiveness of treatment (especially disseminated M.
avium complex infection
PRELIMINARY IDENTIFICATION OF MYCOBACTERIA
 First step is to confirm that the isolate recovered in broth or on
solid media is an acid-fast organism by performing an acid-fast
stain
 Once the organisms are growing on solid media, the following
phenotypic characteristics help speciate mycobacteria:
▪ Colony morphology
▪ Growth rate
▪ Optimal growth temperature
▪ Photoreactivity
COLONY M ORPHOLOGY
 Colonies of mycobacteria are generally distinguished as having
a smooth and soft or rough and friable appearance
 Colonies of Mycobacterium tuberculosis
▪ Rough often exhibit a prominent patterned texture
referred to as cording (curved strands of bacilli)
▪ This texture is the result of tight cohesion of the bacilli
 Colonies of Mycobacterium avium complex (MAC) have a
variable appearance, with glossy whitish colonies often
occurring with smaller translucent colonies
 Optimal temperature and range at which a mycobacterial
species can grow may be extremely narrow, especially at the
time of initial incubation
 M. marinum, M. ulcerans, and M. haemophilum grow best at
30°C to 32°C and poorly, if at all, at 35°C to 37°C
 At the other extreme, M. xenopi grows best at 42°C
PHOTOREACTIVITY
 Mycobacterium spp. have traditionally been categorized into
three groups according to their photoreactive characteristics
 Growth temperature may influence the photoreactive
characteristics of a species
 Photochromogens
▪ Species that produce carotene pigment on exposure to
light
▪ Color ranges from pale yellow to orange
 Scotochromogens – species that produce pigment in the light
or the dark
 Nonchromogenic or nonphotochromogenic
▪ M. tuberculosis is an example
▪ These colonies are a buff (tan) color and are non-
photoreactive
▪ Exposure to light does not induce pigment formation

GROWTH RATE

 Growth rate and recovery time depend on:

▪ Species of mycobacteria
▪ Culture media
▪ Incubation temperature
▪ Initial inoculum size
 Range in recovery time is wide, from 3 to 60 days
 Rapid growers – visible growth in fewer than 7 days
 Slow growers – producing colonies in more than 7 days
 Determination of growth rate should be evaluated from the
time of subculture, not the time of detection from the clinical
sample
 Inoculum should be sufficiently small to produce isolated
colonies
 Microscopic examination of agar for microcolonies allows
earlier detection of growth

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Mycobacteria CHAPTER 25

BIOCHEM ICAL TESTS FOR MYCOBACTERIA  Nitrate reduction test differentiates M. tuberculosis from the
scotochromogens and MAC
 Strongly positive – M. tuberculosis
NIACIN ACCUMULATION  Weakly positive
▪ M. szulgai
▪ M. kansasii
▪ M. fortuitum
▪ M. flavescens

 Most mycobacteria possess the enzyme that converts free

niacin to niacin ribonucleotide
 Accumulation of niacin, detected as nicotinic acid, is the most
commonly used biochemical test for the identification of MTB
 Nicotinic acid reacts with CNBr in the presence of an amine to
form a yellow-pigmented compound CATALASE
 Reagent-impregnated strips have eliminated the need to
handle and dispose of cyanogen bromide, which is caustic and
toxic
 Cyanogen bromide must be alkalinized with NaOH before
disposal
 Niacin test may be negative when performed on young cultures
with few colonies
 Recommended that the test be done on egg agar cultures 3 to 4
weeks old and with at least 50 colonies
 Tests that yield negative results may need to be repeated in
several weeks
 Mycobacteria are catalase-positive
 Test should not be performed on scotochromogenic or rapidly
 However, not all strains produce a positive reaction after the
growing species because M. simiae, bacillus of Calmette-Guérin
culture is heated to 68°C for 20 minutes
(BCG) strain of M. bovis, M. africanum, M. marinum, M.
 Isolates that are catalase-positive after heating have a heat-
chelonae, and M. bovis may be positive, although this occurs
stable catalase
rarely
 Most M. tuberculosis complex organisms do not produce heat-
 Results are most consistent when the test is performed on egg-
stable catalase
based media
 Exceptions are certain strains resistant to isoniazid
 Other heat-stable, catalase-negative species include:
NITRATE REDUCTION ▪ M. gastri
▪ M. haemophilum
 Production of nitrate reductase (nitratase or nitroreductase),
▪ M. marinum
which catalyzes the reduction of nitrate to nitrite, is relatively
uncommon among Mycobacterium spp.
 A positive result may be seen in: HYDROLYSIS OF TWEEN 80
▪ M. kansasii
 Some mycobacteria possess a lipase that can split the
▪ M. szulgai
detergent Tween 80 into oleic acid and polyoxyethylated
▪ M. fortuitum
▪ M. tuberculosis

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Mycobacteria CHAPTER 25

sorbitol pH indicator, neutral red, is initially bound to Tween 80 ACID PHOSPHATASE

and has an amber color
 Acid phosphatase of some mycobacteria separates the
 After hydrolysis of Tween 80, neutral red can no longer bind,
phenolphthalein from magnesium salt thymolphthalein
and it is released, causing a pink color to form
monophosphate
 Time required for hydrolysis is variable – results are recorded as
 Appearance of a red color is positive for acid phosphatase
positive after 24 hours, 5 days, or 10 days
 Mycobacterium tuberculosis is negative for this test
 Helpful in distinguishing between scotochromogenic (positive)
and nonphotochromogenic mycobacteria (negative)

PYRAZINAMIDASE TEST
IRON UPTAKE

 Pyrazinamidase hydrolyzes pyrazinamide to pyrazinoic acid and

ammonia in 4 days
 Some mycobacteria can convert ferric ammonium citrate to an  Ferrous ammonium sulfate combines with pyrazinoic acid,
iron oxide producing a red pigment
 After growth of the isolate appears on an egg-based medium  This reaction occurs in about 4 days
slant, rusty brown colonies appear in a positive reaction on the  May be useful in distinguishing M. marinum from M. kansasii
addition of 20% aqueous solution of ferric ammonium citrate and M. bovis from M. tuberculosis
 Color formation is the result of iron uptake
 Useful in distinguishing M. chelonae, which is generally
negative, from other rapid growers, which are positive
TELLURITE REDUCTION
ARYLSULFATASE
 Most members of the genus Mycobacterium possess the
enzyme arylsulfatase
 This enzyme hydrolyzes the bond between the sulfate group
and aromatic ring structure in compounds
 Tripotassium phenolphthalein sulfate is a molecule, from which
phenolphthalein is liberated with exposure to arylsulfatase
 Liberation of phenolphthalein causes a pH change in the
presence of sodium bicarbonate, indicated by the formation of
a pink color change
 M. fortuitum complex, M. chelonae, M. xenopi, and M. triviale
have rapid arylsulfatase activity that can be detected in 3 days  Reduction of colorless potassium tellurite to black metallic
 M. marinum and M. szulgai exhibit activity with 14 days of tellurium in 3 to 4 days is a characteristic of MAC
incubation  Useful in distinguishing MAC from other nonchromogenic
species
 In addition, all rapid growers are able to reduce tellurite in 3
days

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Mycobacteria CHAPTER 25

UREASE  This is not the same formulation of MacConkey agar typically

used for the isolation of enteric bacilli

GROWTH IN SAUTON AGAR WITH 0.2% PICRIC ACID

 Differentiates slow-growing mycobacteria from rapid-growing
mycobacteria
 Rapid-growing mycobacteria, with exception of M. chelonae,
grow in this medium
 Slow-growing mycobacteria, such as M. simiae, are unable to
grow

 Detection of urease activity can be used to distinguish M. GROWTH IN THE PRESENCE OF P -NITROBENZOIC ACID
scrofulaceum, urease-positive, from M. gordonae, urease-
 p-Nitrobenzoic acid inhibits the growth of M. tuberculosis
negative
complex:
 A loopful of test organism is grown in 4 mL of urea broth at
▪ M. tuberculosis
37°C for 3 days
▪ M. bovis
 Pink to red color is indicative of a positive reaction
▪ M. africanum
 Urease-positive Mycobacterium spp.:
▪ M. microti
▪ M. tuberculosis
▪ M. canettii
▪ M. bovis
▪ M. kansasii
▪ M. scrofulaceum MYCOBACTERIUM TUBERCULOSIS COMPLEX
▪ M. flavescens
▪ M. gastri MYCOBACTERIUM TUBERCULOSIS
▪ M. fortuitum
 Etiologic agent of tuberculosis and Pott’s disease
▪ M. chelonae
 Gram-positive cell wall but are not stained during Gram’s
staining (gram-ghosts)
THIOPHENE-2-CARBOXYLIC ACID HYDRAZIDE (T2H)  Strict aerobe
 T2H distinguishes M. bovis from M. tuberculosis  Tapered ends and may exhibit cording due to elaboration of
 M. bovis is susceptible to lower concentrations of T2H than cord factor
MTB
 Variability in inhibition exists, depending on the concentration
of the inhibitory agent and the temperature of incubation

5% SODIUM CHLORIDE TOLERANCE

 High salt concentration (5% NaCl) in egg-based media (e.g., LJ)
inhibits the growth of most mycobacteria
 M. flavescens, M. triviale, and most rapidly growing
Mycobacterium spp. are exceptions that do grow in the
presence of 5% NaCl

GROWTH ON MACCONKEY AGA R

 Slow growing
 Mycobacterium fortuitum-chelonae complex can grow on  Grows 3-6 weeks at 35°C to 37°C (optimum growth) in the dark
MacConkey agar without crystal violet, whereas most other  No growth at 25°C or 45°C
mycobacteria cannot

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Mycobacteria
 On the other hand, M. marinum, M. haemophilum and M.
ulcerans grow at 30°C to 32°C, while M. xenopi at 42°C
 pH should be 6.5-6.8
 Growth is enhanced by 5-10% CO2
 Cultures are in slanting position with loose caps
 Cultures are examined twice weekly for growth
 Cultures are discarded if no growth after 8 weeks (AFB-negative
smears) or after 12 weeks (AFB-positive smears)
 Colonies are typically raised, with a dry, rough, warty
appearance, non-pigmented and classically described as being
buff-colored
 Colonies may enlarge forming a “cauliflower” center
 Colonies easily detach from the medium
 Niacin positive
 Reduces of nitrate to nitrite (nitrate positive)
 Catalase-positive but heat-stable, catalase-negative
 Isoniazid-resistant strains may not produce catalase at all
 Inhibited by nitroimidazopyran or p-nitroacetylamino-β-
propiophenone (NAP)
 Can be distinguished from M. bovis by:
▪ Inhibition of M. bovis by thiophene-2-carboxylic acid
hydrazide (T2H)
▪ Pyrazinamidase activity
 Appearance of some Mycobacterium spp. in stained smears or
cultures as compared with MTB
▪ M. tuberculosis – slender with beaded or banded
appearance
▪ M. avium complex – coccobacillary forms
▪ M. kansasii – longer, larger, beaded or banded “barber
pole” arrangement
▪ M. ulcerans – produces pale yellow colonies which may be
confused with M. tuberculosis
MYCOBACTERIUM BOVIS
 Produces TB primarily in cattle but also in other ruminants, as
well as in dogs, cats, swine, parrots, and humans
 Disease in humans closely resembles that caused by M.
tuberculosis and is treated similarly
 Primary agent of intestinal TB in man through ingestion of
contaminated milk
 Source of BCG (Bacille Calmette-Guérin) vaccine
 Grows very slowly on egg-based media, producing small,
granular, rounded, non-pigmented colonies with irregular
margins after 21 days of incubation at 37°C
 On Middlebrook 7H10 medium, colonies are similar to those of
M. tuberculosis but slower to mature
CHAPTER 25
 Most strains of M. bovis are:
▪ Niacin-negative
▪ Do not reduce nitrate
▪ Do not grow in the presence of T2H
 The above biochemical characteristics distinguish M. bovis from
most strains of M. tuberculosis
MYCOBACTERIUM LEPRAE
 Hansen’s bacillus
 Etiologic agent of Hansen’s disease or leprosy – an infection of
the skin, mucous membranes, and peripheral nerves
 Hansen disease is not considered highly contagious
 The two major forms of the disease are:
▪ Tuberculoid leprosy – skin lesions with loss of sensation;
patients exhibit an effective CMI response
▪ Lepromatous leprosy - slowly progressive, life-threatening
if untreated; characterized by skin lesions and progressive,
symmetric nerve damage; do not produce an effective CMI
response
 In patients with tuberculoid leprosy, organisms are extremely
rare and may not be detected in skin scrapings or biopsy
specimens
 However, AFB are usually abundant in samples from patients
with lepromatous leprosy
 Morphologically similar to M. tuberculosis
 Exhibits cigar pack, picket fence, or palisade arrangement
 Smears positive for M. leprae should contain both AFB and
Lepra cells, large cells with uniform staining
 Cannot grow in artificial media, thus grown in armadillos and
footpad and earlobe of mice
Lepra cells. Large cells with their abundant cytoplasm, occupying the
greater part of the cell and filled with mycobacteria leprae bacilli,
highlighted by modified Fite Faraco (MFF stain, x1000)
 Definitive laboratory diagnosis is the development of disease in
laboratory mice following inoculation of patient biopsy material
to the mouse footpad
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Mycobacteria CHAPTER 25

SLOWLY GROWING SPECIES  Most strains are strongly catalase-positive (>45 mm in

semiquantitative test)
MYCOBACTERIUM AVIUM COMPLEX  Strains that are low-catalase producers (<45 mm) are less
commonly isolated
 Mycobacterium avium and M. intracellulare are part of the  Characteristics that distinguish this species are:
Mycobacterium avium complex (MAC) ▪ Growth rate similar to that of M. tuberculosis at 37°C
 High prevalence in patients with AIDS ▪ Strong photochromogenic properties
 More than 90% of MAC organisms that are isolated from AIDS ▪ Ability to hydrolyze Tween 80 in 3 days
patients are Mycobacterium avium – distinguished from M. ▪ Strong nitrate reduction, catalase production, and
intracellulare by NAAT pyrazinamidase production
 Mode of transmission is through the GIT via infected semen
 Initial symptom is protracted diarrhea
 Pulmonary disease is common in elderly and in
immunocompromised patients
 Because the two species in the MAC are so similar, most
laboratories do not distinguish between them but report
isolates of both species as MAC
 On primary isolation media, these organisms grow slowly,
producing thin, transparent or opaque, homogeneous smooth
colonies
 A small proportion of strains may exhibit rough colonies
 Usually the colonies are non-pigmented, but they may become
yellow with age
 Rarely are the colonies pigmented from the onset of detectable
growth
 Optimal growth temperature is 37°C MYCOBACTERIUM HAEMOPHILUM
 On microscopic examination, the cells are short, coccobacillary,
 Occur primarily in patients who are immunocompromised with
and uniformly stained, without beading or banding
cases reported in patients with Hodgkin disease and AIDS
 Long, thin, beaded bacilli resembling Nocardia spp. may be seen
 Unique characteristic of this organism is its requirement for
in stains of very young cultures or under certain other
hemoglobin or hemin for growth
conditions
 Isolation is accomplished on media supplying the needed
 MAC species are inactive in most physiologic tests used to
growth supplement, such as:
identify the mycobacteria
▪ Chocolate (CHOC) agar
 Exceptions are the production of a heat-stable catalase and the
▪ Mueller-Hinton agar with 5% Fildes enrichment
ability to grow on media containing 2 μg/mL of T2H
▪ Löwenstein-Jensen (LJ) medium containing 2% ferric
ammonium citrate
MYCOBACTERIUM KANSASII  Successful isolation on Middlebrook 7H10 agar with an X factor
disk planted in the inoculated area has been reported
 Second to MAC as the cause of NTM lung disease  Optimal growth temperature is 28°C to 32°C
 Slow-growing organism that appears as long rods with distinct  Little or no growth occurs at 37°C
crossbanding  Colonies are rough to smooth and non-pigmented
 M. kansasii has an optimal growth temperature of 37°C, and  Microscopically, cells are strongly acid-fast, short, occasionally
colonies appear smooth to rough, with characteristic wavy curved bacilli without banding or beading, and arranged in tight
edges and dark centers when grown on Middlebrook 7H10 agar clusters or cords
 Some cording can usually be seen with low-power
magnification MYCOBACTERIUM MARINUM
 Colonies are photochromogenic
 Cutaneous infections in humans occur when traumatized skin
 With prolonged exposure to light, most strains form dark red
comes into contact with salt water or inadequately chlorinated
crystals of β-carotene on the surface of and inside the colony
fresh water containing the organism

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Mycobacteria
 Tender red or blue-red subcutaneous nodule, or swimming
pool granuloma, usually occurs on the elbow, knee, toe, or
finger
 Cells of M. marinum are moderately long to long rods with
cross barring
 Colonies of this slowly growing organism are smooth to rough
and wrinkled on inspissated egg medium but may be smooth
when grown on Middlebrook 7H10 or 7H11 agar
 Photochromogenic
▪ Young colonies grown in the dark may be non-pigmented
or buff
▪ Colonies grown in or exposed to light develop a deep
yellow color
 Growth is optimum at incubation temperatures of 30°C to 32°C
 Clues to identification of M. marinum:
▪ Growth at lower incubation temperatures
▪ Photochromogenic
 Some strains of M. marinum produce niacin
 Nitrate negative
 Heat-stable catalase negative
 Hydrolyze Tween 80
 Urease positive
 Pyrazinamidase positive
MYCOBACTERIUM SCROFULACEUM
 Most common form of disease associated with M. scrofulaceum
is cervical lymphadenitis in children
 On microscopic examination, uniformly stained, acid-fast,
medium to long rod
 Grows slowly (4 to 6 weeks) at incubation temperatures ranging
from 25°C to 37°C
 Colonies are smooth with dense centers and pigmentation from
light yellow to deep orange
 The organism is scotochromogenic
 Do not hydrolyze Tween 80 or reduce nitrate
CHAPTER 25
 Produce urease and are high (>45 mm) catalase producers
 These characteristics aid in differentiating this organism from
other slow-growing scotochromogens, including certain strains
of MAC, M. gordonae, and M. szulgai
MYCOBACTERIUM SIMIAE
 Appear as short coccobacilli
 When they are grown on inspissated egg medium at 37°C,
smooth colonies appear in 10 to 21 days
 Colonies on Middlebrook 7H10 agar are thin, transparent or
tiny, and filamentous
 Usually photochromogenic
 Development of the yellow pigment may require prolonged
incubation, whereas some strains may fail to produce pigment
on exposure to light
 Differential biochemical characteristics are:
▪ Niacin positive
▪ Negative nitrate reduction
▪ Production high-level, heat-stable catalase
 M. simiae is one of the very few NTM that produces niacin –
may be identified as M. tuberculosis if pigment production
under light is not observed
MYCOBACTERIUM ULCERANS
 Rare cause of mycobacteriosis, also referred to as Buruli ulcer
 Third most common Mycobacterium spp., behind M.
tuberculosis and M. leprae
 The disease manifests as a painless nodule under the skin after
previous trauma
 A shallow ulcer develops that may be quite severe
 Acid-fast cells of M. ulcerans are moderately long, without
beading or crossbanding
 Optimal growth temperature is 30°C to 33°C, with little growth
at 25°C and usually none at 37°C
 Organism grows slowly, often requiring 6 to 12 weeks of
incubation before colonies are evident
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Mycobacteria
 Colonies are smooth or rough and non-pigmented or lightly
buff, and they do not develop pigment with exposure to light
 M. ulcerans produces a heat-stable catalase but is inert in most
other conventional biochemical tests
MYCOBACTERIUM XENOPI
 On acid-fast–stained smears, M. xenopi are long filamentous
rods
 Colonies on Middlebrook 7H10 agar are small, with dense
centers and filamentous edges
 Microscopic observation (LPO) of colonies growing on
cornmeal-glycerol agar reveals distinctive round colonies with
branching and filamentous extensions
 Aerial hyphae are usually seen in rough colonies
 Furthermore, young colonies grown on cornmeal agar have a
bird’s nest appearance, with characteristic sticklike projections
 Distinctive characteristics:
▪ Optimal growth at 42° C – grows more rapidly at this
temperature than at 37°C and fails to grow at 25°C
▪ Yellow scotochromogenic pigment – classified with
nonphotochromogenic group; however, colonies are
frequently bright yellow on primary isolation when
incubated in the absence of light and when exposed to
light
▪ Negative reactions for niacin accumulation, nitrate
reduction
▪ Positive reactions for the production of heat-stable
catalase, arylsulfatase, and pyrazinamidase
RAPIDLY GROWING SPECIES
MYCOBACTERIUM CHELONAE -MYCOBACTERIUM
ABSCESSUS GROUP
 Three most important rapidly growing mycobacteria causing
human infections are:
▪ M. abscessus subsp. abscessus (formerly M. abscessus)
▪ M. chelonae
▪ M. fortuitum
 Young cultures of M. chelonae are strongly acid-fast, with
pleomorphism ranging from long, tapered to short, thick rods
 This rapidly growing mycobacterium produces rough or
smooth, non-pigmented to buff colonies within 3 to 5 days of
incubation at 37°C
 Characteristics that help differentiate M. chelonae and M.
abscessus from other nonchromogenic, rapidly growing
mycobacteria:
CHAPTER 25
▪ Positive 3-day arylsulfatase test
▪ No reduction of nitrate
▪ Growth on MacConkey agar without crystal violet
MYCOBACTERIUM FORTUITUM GROUP
 M. fortuitum group is the most common rapidly growing
mycobacteria associated with localized cutaneous infections
 After 3 to 5 days of incubation at 37°C, colonies of M. fortuitum
appear rough or smooth and non-pigmented, creamy white, or
buff
 Microscopic examination of growth on cornmeal-glycerol and
Middlebrook 7H11 agars after 1 to 2 days of incubation reveals
colonies with branching filamentous extensions and rough
colonies with short aerial hyphae
 On microscopic examination, cells are pleomorphic, ranging
from long and tapered to short, thick rods
 Cells from most cultures, especially older ones, tend to
decolorize and appear partially acid-fast with any of the acid-
fast staining techniques
 Additional characteristics that distinguish M. fortuitum from
other rapidly growing mycobacteria are:
▪ Positive 3-day arylsulfatase test
▪ Reduction of nitrate
MYCOBACTERIUM SMEGMATIS GROUP
 M. smegmatis group contains two species:
▪ M. smegmatis
▪ M. goodie
 Commonly considered saprophytic, M. smegmatis has been
implicated in rare cases of pulmonary, skin, soft tissue, and
bone infections
 On acid-fast stain, cells are long and tapered or short rods with
irregular acid fastness
 Occasionally rods are curved with branching or Y-shaped forms;
swollen, with deeper staining, beaded, or ovoid forms are
sometimes seen
 Colonies appearing on egg medium after 2 to 4 days are usually
rough, wrinkled, or coarsely folded; smooth, glistening,
butyrous colonies may also be seen
 Colonies on Middlebrook 7H10 agar are heaped and smooth or
rough with dense centers
 Pigmentation is rare or late
 Colonies appear non-pigmented, creamy white, or buff to pink
in older cultures
 In addition to the rapid growth rate and non-pigmented rough
colonies, characteristics valuable in the identification of this
organism are:
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Mycobacteria CHAPTER 25

▪ Negative arylsulfatase reaction

▪ Positive iron uptake MIDDLEBROOK 7H10 AND 7H11 AGARS
▪ Ability to reduce nitrate
▪ Growth in the presence of 5% NaCl
▪ Growth on MacConkey agar without crystal violet

APPENDIX

SELECTED CULTURE MEDIA

LÖWENSTEIN -JENSEN (LJ) MEDIUM

Purpose:
▪ Used to cultivate Mycobacterium spp.
▪ Isoniazid-resistant strains grow better on these media,
especially Middlebrook 7H11, than on egg-based media
such as LJ
▪ Middlebrook agars are also more chemically defined than
the LJ formulations

Principle:

Purpose:
▪ Used to cultivate Mycobacterium spp.
Principle:
▪ Potato flour, egg, and glycerol help detoxify this medium
and also supply nutrients required for growth of these
organisms
▪ Asparagine is included for maximum production of niacin
by certain Mycobacterium spp.
▪ Malachite green inhibits the growth of other bacteria that
may be present in specimens
▪ LJ medium must be kept out of direct light because
malachite green is light- sensitive
▪ Middlebrook 7H10 and 7H11 are similar, except 7H11
contains casein hydrolysate, which stimulates the growth
of drug-resistant Mycobacterium tuberculosis
▪ Both media contain growth factors, such as amino acids,
glycerol, and inorganic salts that encourage recovery of
mycobacteria
▪ In addition, both formulations include OADC (oleic acid-
dextrose-catalase) enrichment, which chemically simulates
egg components
▪ Oleic acid is a fatty acid used by the mycobacteria
▪ Albumin – inhibit toxic agents that might be present and
provide a source of protein
▪ Dextrose – used by the mycobacteria to generate energy
▪ Exogenous catalase neutralizes toxic peroxides
▪ Malachite green is also added, although at a lower
concentration than in LJ, and it gives some selectivity to
the medium

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Mycobacteria CHAPTER 25

MITCHISON 7H11 SELECTIVE AGAR

Purpose:
Selective agar for isolation of Mycobacterium spp.

Principle:
Prepared by adding antimicrobial agents to the Middlebrook 7H11
formulation, thereby making the medium more selective for
mycobacteria
Amphotericin B, carbenicillin, polymyxin B, and trimethoprim are
incorporated to make Mitchison 7H11 selective agar more inhibitory
to gram-negative rods in particular, as well as yeast

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