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Morphological Characterization of Mycobacterium

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DOI: 10.5772/29644 · Source: InTech

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 8

Morphological Characterization of
Mycobacterium tuberculosis
Ali Akbar Velayati and Parissa Farnia
Mycobacteriology Research Centre,
National Research Institute of Tuberculosis and Lung Disease (NRITLD),
WHO & UNION Collaborating Centre for TB & Lung Diseases,
Shahid Beheshti University (Medical Campus), Darabad, Tehran,
Iran

1. Introduction
It is more than 100 years since the first Mycobacterium was isolated by Hansen (1874). That
was leprosy bacillus, Mycobacterium leprae, which even today is still resisting all attempts to
cultivate it in the laboratory. The tubercle bacillus, M. tuberculosis was discovered eight years
later by Robert Koch (1882). The Koch discovery was confirmed by more efficient staining
models of Ehrlich (1887) and Ziehl- Neelsen (1883). Under Light microscope, the tubercle
bacilli typically appear as straight or slightly curved rods. According to growth conditions
and age of the culture, bacilli may vary in size and shape from coccobacilli to long rods. The
dimensions of the bacilli have been reported to be 1-10 µm in length (usually 3-5 µm), and
0.2 -0.6 µm width. The possibility of morphological variations in tubercle bacilli was
suggested by few investigators like Malassez and Vignal (1883), Nocard and Roux (1887),
Metschnikoff (1888), Lubarsch(1899), Fischel(1893), and Vera and Rettger (1939). They
showed under unfavorable conditions, i.e., a limited food supply, or oxygen deprivation,
Mycobacterium assumed a swollen appearance without forming the vacuolar or globoid
bodies (Vera and Rettger, 1939). These early reports were based on stained preparations and
were subjected of severe criticism (Porter and Yegian, 1945). Today with advances in
microscopic technique i.e., transmission electron microscope (TEM), scanning electron
(SEM) and atomic force microcopy (AFM), almost all of investigators have been agreed that
the Koch bacillus does not always manifest itself in the classical rod shape (figure 1). They
become shorter in older cultures, filametous within macrophages and ovoid during
starvation (Young et al., 2005; Farnia et al., 2010; Shleeva et al., 2011) and they may produce
buds (Chauhan et al., 2006) and branches in extensively drug resistance strains (XDR-TB)
(Velayati et al 2010; Farnia et al 2010). In the following parts the underlying mechanisms that
may help the bacilli to change its morphology was highlighted.

2. The role of cell wall in shape maintenance

The cell wall of mycobacterium is characterized by a unique structure which is caused by
partly distinct chemical compositions in comparison with the cell wall of other bacteria
150 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

(Koike and Takeya, 1961; Imaeda and Ogura, 1963; Imaeda et al., 1969). These variations are
thought to be advantageous in stressful conditions of osmotic shock or desiccation as well as
contributing to their considerable resistance to many drugs (Jarlier and Nikaido, 1990). The
Mycobacterial cell wall, in principal, consists of an inner layer and an outer layer that
surround the plasma membrane (Hett and Rubin, 2008). The outer compartment consists of
both lipids and proteins (Draper, 1971, 1998; Draper et al., 1998; Brennan and Nikaido, 1995;
Brennan, 2003). The inner compartment consists of peptidoglycan (PG), arabinogalactan
(AG), and mycolic acid (MA) covalently linked together to form a complex known as MA-
AG-PG complex that extends from the plasma membrane outward in layers, starting with
PG and ending with MAs. The Peptidoglycan, which forms the “backbone’ of the cell wall
skeleton,was first studied by Misaki et al (1966). It belongs to a family of structures
possessed by almost all bacteria and blue-green algae but by no other type of living
organism (Schleifer and Kandler, 1977); its presence in mycobacteria provides conclusive
evidence that they are not, as was once believed, some sort of intermediate stage between
bacteria and fungi. The peptidoglycon is made of peptides and glycan strands. The long
glycan strand typically consists of repeating N-acetylglucosamines (NAGs) linked to
N-acetylmuramic acid (NAM). These strands are cross linked by peptides bound to the
lactyl group on NAMs from different glycan strands. These peptide chains normally consist
of L-alanyl-D-iso-glutaminyl-meso-diaminopimelic acid (DAP) from one strand linked to the
terminal D-alanine residue from L-alanyl-D-iso-glutaminyl-meso- DAP-D-alanine from a
different strand (Kotani et al., 1970; Wietzerbin et al., 1974). This highly cross-linked glycan
meshwork of PG that surrounds bacteria is the primary agent that maintains bacterial shape.
The structure of this stratum differs slightly from that of common bacteria, as it presents
some particular chemical residues and unusual high number of cross-links. Indeed, the
degree of peptidoglycon cross linking in the cell wall of M. tuberculosis is 70-80%, whereas
that in E. coli is 20-30%. (Matsuhashi, 1994; nVollmer and Holtje,2004). PG isolated from
E. Coli retains its rod –like shape even in the absence of all other material (Weidel et al., 1960;
Weidel and Pelzer, 1964), confirming its role in shape maintenance. Also, treatment of
bacteria with lysozyme which degrades PG, results in rod shaped cells becoming round
spheroplasts (Lederberg, 1956). Spheroplasts, or round bacteria lacking PG, can be formed in
M. smegmatis through degradation of PG. Upon transfer to growth media, the spherical
bacteria are able to regenerate wild-type rod -shaped cells (Udou et al., 1982). This occurs
through elongation of bacteria that then branch, septate and fragment. These data argue that
shape and size are not simply governed by existing PG, but there must be some genetic
heritable determinant also.

3. Peptidoglycan synthesize
Little is known about the biosynthesis of the peptidoglycan of M. tuberculosis. However, it is
generally assumed to be similar to that of E. coli (van Heijenoort, 1998). Generally,
peptidoglycan synthesis occurs in four sequential steps. First, inside the cytoplasm, soluble
substrates are activated and peptidoglycon units are build. Glucosamine is enzymatically
converted into MurNAc and then energetically activated by a reaction with uridine
triphosphate (UTP) to produce uridine diphosphate –N-acetylmuramic acid (UDP-MurNAc)
(De Smet et al., 1999). Second, at cytoplasmic membrane, the units UDP-MurNAc
pentapeptide is attached to the bactoprenol “ conveyor belt”, through a pyrophosphate link
Morphological Characterization of Mycobacterium tuberculosis 151

with the release of uridine monophosphate (UMP)(Crick et al., 2001; Yuan et al., 2007). Third
the bactoprenol molecule translocates the disaccharide pentapeptide precursor to the
outside of the cell. The GlcNAc-MurNAc disaccharide is then attached to a peptidoglycan
chain using pyrophosphate link between itself and the bactoprenol as energy to drive the
reaction. The pyrophosphobactoprenol is converted back to a phosphobactoprenol and
recycled. Fourth, outside the cell but near the membrane surface, peptide chains from
adjacent glycan chains are cross-linked to each other by a peptide bond exchange
(transpeptidation) between the free amine of the amino acid in the third position of the
pentapepide (e.g., lysine) or the N-terminus of the attached pentaglycine chain and the
D-alanine at the fourth position of the other peptide chain, releasing the terminal D-alanine
of the precursor (Wietzerbin et al., 1974; Ghuysen, 1991).

4. Control of peptidoglycan synthesis

Enzymes involved in remodeling PG can be grouped as either biosynthetic or hydrolytic.
Biosynthetic enzymes include transglycosylase and transpeptidase domains, often found on
a single, bifuntional protein. Hydrolytic enzymes include muramidase, glucosaminidase,
lytic transglycosylase, amidase, endopeptidase and carboxypeptidase (Young, 2003; Cabeen
and Jacobs-Wagner, 2005). The reaction of these enzymes may be antagonistic, or they may
physically interact to form complexes capable of breaking bonds to generate openings for
new monomers, while also forming bonds necessary to unit PG strands. The production of
these enzymes should be regulated, otherwise the bacterial cell wall would be degraded and
the bacteria would be lyses. There are several ways to governorate these enzymes; one
method is through formation of complexes with other proteins (Hett and Rubin, 2008).These
proteins could suppress the activity of the enzyme, or they could be enzymes themselves
with antagonistic reactions that join rather than degrade PG. Another possibility is that the
enzymes are sequestered from their substrate until they needed. A third method could be
that the appropriate substrate is not made available until cleavage of it is required.

5. The role of PG in cell shape regulation

The PG –synthesizing enzyme organize into complexes that likely contributes to the
resulting shape. Various models have been proposed which explain how this organization
affects the bacterial shape. The two –competing sites” model (TCS) for peptidoglycan
assembly advocates that, in bacterial rods, the shape depends on the activity of two
biochemical reactions (sites) which occur in the terminal stages of peptidoglycan synthesis;
one site is responsible for lateral wall elongation , and the other is responsible for septum
formation (Lleo et al., 1990; Alaedini and Day, 1999). The two sites compete with each other
in such a way that the lateral wall is not extended during septum formation and vice versa
(Lleo et al., 1990; Satta et al .,1994). The actual shape of the bacteria is thus determined by the
balance between the two competing reactions, correct balance leading to normal rods;
abnormal prevalence of the site for lateral wall elongation leads to long rods or filaments,
whereas prevalence of the site for septum formation leads to formation of coccobacilli or
cocci (Lleo et al., 1990). The other bacteria carry only one site for peptidoglycan assembly
which can form only septa and can grow only as cocci. Another model is “three –for –one “
predict the insertion of PG along a track, using an existing strand of PG as a template
152 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

(Holtje, 1998). This result in doubling the length in one direction, but since following a
strand, no additional length is added in the direction perpendicular to the strand. Thus
width would stay constant. Another theory as to how cells maintain a constant width posits
that the poles are capped with a type of PG that prevents rapid turnover or insertion of new
PG (De Pedro et al., 1997). Thus, the caps would restrict the width of the bacterium

6. How the shape remain constant

Uniform cell shapes are favored by the need to segregate the chromosome and cytoplasmic
material between daughter cells (Errington et al., 2003). The regular shape would seem to be
the best way to ensure each daughter, because a symmetrical cell can be halved accurately
by mechanisms that measure length or volume (Helmstetter et al., 1990; Young, 2006). In an
irregular cell, misplaced septation might leave one cell with both chromosomes or with
more than its fair share of other components. Therefore, once a particular shape is adapted
bacteria have a vested interest in keeping it (Stewart, 2005). The major incentive for doing so
is to maintain a consistent relationship between cytoplasmic volume and surface area so that
cell cycle events can be coordinated properly. This is visualized by considering the septation
event that creates two daughter cells (Harry, 2001; Errington et al., 2003). The septum is
formed through the in-ward growth of cytoplamic membrane and cell wall material that
invaginates from opposing directions at the central plane of the cell. In such case, the
concentration of essential division proteins will not change, but the surface area over which
they must act will be greater in the sphere. The amounts of these proteins, if optimized for
dimensions of a rod , might not be sufficient to initiate or complete normal septation and
division in a coccus (Young, 2006). Thus limited concentrations of division proteins will
dictate that the cell maintain a specific and constant diameter. To do this, bacteria must
coordinate events spatially and temporally. Recently it was shown that the divisome will
assemble at midcell, before chromosomes partitioned. The divisome consists of a set of 10 to
15 proteins that are required to the middle of the cell and are responsible for generating the
septum that divides two daughter cells (Margolin, 2006; Buddelmeijer and Beckwith, 2002).
This is accomplished by synthesizing septal PG, constricting the cell wall to eventually close
off the cytoplasmic compartments of each daughter cell, and finally hydrolyzing part of the
PG that holds two together in order to physically separate the cells. These divisome proteins
(FtsA, FtsB, FtsE, FtsI, FtsK, FtsL, FtsN, FtsQ, FtsW, FtsX, FtsZ, Zip A, AmiC and EnvC)
encoded in different bacterial genomes and have different function (Di Lallo et al., 2003;
Karimova et al., 2005; Vicente and Rico, 2006). The FtsZ is the first protein to assemble at
midcell (Bi and Lutkenhaus, 1992). Its formation of a ring around the cell, just under the
plasma membrane, gives the assembled divisome the name Z ring. This sub cellular
organelle, a functional analog of the contractile ring used in cytokinesis of many eukaryotic
cells, is thought to form the scaffold for recruitment of the other key cell division proteins. In
E. coli, successful cell division depends on a constant and critical concentration of Ftsz
combined with proper proportions of Z-ring stabilizing and destabilizing proteins.
Significantly, small changes in the concentrations of FtsZ or other essential division proteins
disrupt cell growth. Thus, division is inhibited if FtsZ is under produced, extra divisions
occur if the protein is overproduced and no division occurs if FtsZ levels are adequate but
FtsZ/FtsA ratio is incorrect (Errington et al., 2003; Maki et al., 2000; Chauhan et al., 2006).
Morphological Characterization of Mycobacterium tuberculosis 153

7. Shape variation
The tubercle bacillus is a prototrophic (i.e., it can build all its components from basic carbon
and nitrogen sources) and heterotrophic (i.e., it uses already synthesized organic
compounds as a source of carbon and energy), metabolically flexible bacterium( Edson, 1951;
Ramakrishnan et al., 1972; Niederweis, 2008). The success of tubercle bacilli as a pathogen
can be attributed to its extraordinary capacity to adapt to environmental changes
throughout the course of infection. Generally, the nutritional quality and physical
conditions will determine the temporary lifestyle of bacillus. These changes include:
nutrient deprivation, hypoxia, temperature, PH, salinity and various exogenous stress
conditions (Vera and Rettger, 1939; Smeulders et al., 1999; Honer et al., 2001; Young et al,
2005; Anuchin et al., 2009; Velayati et al, 2009; Farnia et al., 2010; Singh et al., 2010; Shleeva et
al., 2002, 2010). Unfortunately, in most of cases we do not know if shape per se is beneficial,
because few experiments have addressed the question. Knowledge of the physiology of M.
tuberculosis during this process has been limited by the slow growth of the bacterium in the
laboratory and other technical problems such as cell aggregation. Recent advances in
microscopy techniques have revealed adaptive changes in size and shape of bacilli under

Fig. 1. Scanning electron microscope shows shape variation in M. tuberculosis at exponential

phase of growth.
154 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

stress conditions (Velayati et al., 2009,2011; Farnia et al., 2010). Briefly, the reported
morphological variation in M. tuberculosis are classified into two categories; those which
frequently seen at exponential phase of growth that is rod, V, Y-shape, branched or buds,
and those that are seen occasionally under stress or environmental conditions which are
round, oval , ultra-virus, spore like, and cell wall defiant or L-forms.

8. Shape variations during active or exponential phase of growth

The most classical form of tubercle bacilli is a slender rod shape that seen in stained smears.
They have smooth, homogenous cytoplasm with clear-cut and well-define outlines. The first
electron microscope images of the tubercle bacilli were obtained in 1939 in the laboratories
of the Technische Hochschule, Berlin.
von Borries and E. Ruska (1939) published electron micrographs of the avian strain of
tubercle bacilli magnified 26,000 times. The cytoplasm of these bacilli contained dark bodies
of different sizes. Later on, Lembke and Ruska (1940), culture the bacilli on petragnani
medium and observed up to eight large bodies inside the cytoplasm of bacilli. Rosenblatt,
Fullam and Gessler (1942) in their studies of tubercle bacilli in the electron microscope,
confirmed many earlier observation and added some new data, particularly concerning the
internal structure of bacilli. The bacilli varied in size. The size of the strain H37 sub-cultured
at Columbia University varied from 4.3µ X 0.4µ to 1.0µ X 0.2µ. The cell wall was always
present (sometimes it was as thick as 0.03µ) and contained granules. The internal structure
showed dense nuclear masses within the granular cytoplasm. The density of the cytoplasm
varied; it contained many granules and vacuoles of different sizes. Later on it became clear
that the cytoplasm of young cells is dense, the basic dyes stain it deeply and uniformly, and
it contains vacuoles and hyper chromic bodies. The cell protoplast was seen surrounded by
a 0.023µ thick and ductile cell wall. The cytoplasm itself was covered with a thin
cytoplasmic membrane which closely adhered to the cell wall (Rosenblatt et al., 1942; Knaysi
et al., 1950; Werner, 1951; Draper, 1982). In rod like bacilli, the process of cell division
resembles that of most grams –positive bacteria (figure, 2). In the equatorial zone of the cell,
on the inner side of the cell wall, a double cell plate was formed. The growth of this plate
proceeded till the mother cell wall was divided into two daughter cells. The separation of
newly formed cells occurred between these plates, which then covered the poles of the right
and left cells. Before the cytoplasm divided, the division of cellular bodies was observed
(Edwards, 1970; Nishiura et al, 1970; Dhal, 2004).
The other types of cell shape (V or Y - shape bacilli) occurs in lower frequency (Dahl, 2004;
Farnia et al 2010). The V-shape bacilli are caused by snapping post-fission movements
(Krulwich and Pate, 1971). The term “snapping division” was first described by Kurth (1898)
and has been reported by many other investigators. Upon completion of cell division, one or
both of the two daughter cells suddenly swing around, bringing their distal ends closer
together while still remaining attached by a small region at their proximal ends. The exact
mechanism responsible for snapping postfission movements is not clear. Bisset (1955)
claimed that all so-called postfission movements were nothing but artifacts due to
mechanical stress on the dividing cells (e.g., cells growing between solid agar and a cover
slip) and would not occur if the same cells were grown in liquid cultures. Sguros (1957)
Morphological Characterization of Mycobacterium tuberculosis 155

suggested that V-forms resulted from “germ tube extrusions” from each of a pair of attached
arthrospores and were not due to postfission movements. More studies have demonstrated
that snapping division or V-forms could arise by any of three methods: (I) germination of
adjacent coccoid elements, (ii) subpolar germination (budding) of rods, and (iii) snapping
postfission movements (Starr and Khan, 1962). In mycobacterium, during septum formation
the plasma membrane and inner cell wall grow inward but the outer cell wall layer remains
intact. Upon completion of septum formation with a cross-wall, the inner layer may
continue to grow and thus exert pressure upon the outer cell wall layer. The outer layer
eventually ruptures first on one side of the cell, and the two daughter cells bend in on the
side where the outer layer is still intact forming a “V-form (Dahl,2004; Farnia et al,2010;
Malhotra et al., 2010)
Mycobacterium is known to form a “Y-shaped “cells with branches more interior to the cells
and of greater length figure 3. Brieger et al in 1954, was among the first scientist who
demonstrate the branching in the reproductive cycle of M. avium. He showed that young
culture of bacilli when first transplanted to fresh medium it consists mainly of short
coccoid rods. These elongate into filaments (8-10µ) which continue to divide and grow
during a phase of filamentous proliferation. The filaments usually have two fully

Fig. 2. Atomic force microscopy shows the V-shape M. tuberculosis during exponential phase
of growth
156 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

developed dense bodies in polar positions and in some organisms a number of smaller are
also seen scattered among the cellular units and apparently associated with them. The final
stage in the reproductive cycle led to a massive production of small rods. At this phase the
filaments suddenly break down into masses of short rods which elongate to form the new
generation and the cycle is complete. Under electron microscope, it was seen that the
filaments were quite separate, and there was no true branching and that the mycelia
appearance was produced because the filaments often remained stuck together. In another
study, Mizuguchi Y et al (1985) showed β-Lactam antibiotics at low concentration induced
filamentous cells in the M. avium-intracellular complex. Although, the mechanisms of
induction of filamentous cells appeared to be different according to the drugs used.
Ampicillin induces filaments by inhibiting the septation in a manner similar to its effect on
E. coli, whereas cephazolin induces filaments but does not inhibit septation. In
M. tuberculosis, branches were first seen as a small bud that does not grow to any
appreciable size before breaking off as a separate cell. Few studies suggested that M.
tuberculosis grows from the ends of bacilli and not along the length of the cylinder as seen in
other well-characterized rod shape bacteria (Thanky et al., 2007). This might be true for
susceptible isolates, but recently Farnia et al (2010) showed that in highly drug resistance
strains i.e., XDR-TB and Totally or Extremely drug resistant isolates (TDR or XXDR-TB),
branches produce along the cylinder. In fact, about 20 -24% of cells in XDR and XXDR-TB
bacilli were dividing by branching, respectively.

Fig. 3. Transmission Electron Microscopy shows Y-Shape M. tuberculosis at exponential

phase of growth

9. Cell shapes during dormancy or under limited conditions

The morphological variations in tubercle bacilli become evident when the culture medium
was poor. These changes were first reported by Koch himself. In his paper on the “discovery
Morphological Characterization of Mycobacterium tuberculosis 157

of the cause of tuberculosis”, he described that “under certain conditions, some bacilli
contain several spores, in most cases there are two to four of them; oval in form, they are
distributed, in uniform intervals, along the axis of the bacilli(1882). Following Koch
discovery, Malassez and Vignal (1883) had described, the small “coccoid bodies “ which
cause tuberculosis infection and named them as cell wall deficient forms (CWD-forms) of
tuberculosis. Later on, Spengler (1903, 1905), were among the first scientist who could
demonstrated that in older cultures and frequently in sputa, apparently in response to
adverse environmental conditions, the smooth cell takes on a fragmented appearance. Much
(1907) was able to reproduce granules in the inside of the bacilli as well as scattered around
them. These granules, according to Much, cannot be stained by the Ziehl- Neelsen technique
but may generate new tubercle bacilli. Later on 1909, Fontes revealed how he had applied
double staining to the bacilli, namely Ziehl- Neelson’s carbolfuchsin staining and the Gram
treatment. In this way he tried to differentiate the pathogenic tubercle bacilli, containing
Much granules, from the apathogenic ones without these granules. In 1910, Fontes described
the multiplication through division of these granules in the inside of a cell and on its outside
and applied the term “virus” to this formation. Fontes described the application to the
tubercule bacillus of the well –known method of separating the virus from the substrate by
filtering the material through a bacterial filter. He inoculated a guinea pig with the filtered
caseous material and transplanted the organs of this animal into a fresh one. When after five
months of observation the animal was killed, the autopsy revealed the infiltration of round
cells, granules, and occasional acid-fast bacilli in the lymph nodes and the lungs. After years
of oblivion, the early works of Fontes were rediscovered by Vandremer (1923). He repeated
the Fontes filtration experiments and confirmed the development of acid –fast bacilli on
media and in animals inoculated with these filtrates. Calmette (1926) advanced the theory
on the role of the the tuberculosis ” ultra-virus” in the development of certain forms of the
diseases. However, Negre et al (1933) denied the existence of filterable forms of the
mycobacteria. Few years later, Vera and Rettger (1939) studied four strains of
M. tuberculosis(hominis), “Koch”, 607, 75 and H37 in micro-culture by Hill hanging block
technique. This method was employed to permit observation of individual cells and their
progeny over long periods of time using lucida drawings camera. They could demonstrate
various forms which have been described in the literature at one time or another. When they
cut off air supply, different variants developed very soon. The bacilli swelled slightly, the
cytoplasm become less clear and smooth. The swelling commonly occurred at the ends of
cells, so the clubs and dumbbell shapes were formed; cells often became spoon shaped.
These swollen structures became increasingly refractive and more sharply delimited, until
finally there was a definite superficial resemblance to spores. At the similar time, the ability
of the tubercle bacillus to survive environmental hardship in culture was documented by
Corper and Cohn in a study published in 1933. In another study, McCune and other
colleagues (1965, 1966), showed the capacity of tubercle bacilli to survive in mouse tissue
after sterilization. In this model, out bred mice were infected intravenously with 105 colony-
forming units of the H37Rv strain of M. tuberculosis. They were immediately treated for a
period of 12 weeks with the antimycobacterial drugs isonizid (INH) and pyrazinamide
(PZA). For 4-6 week period after withdrawal of therapy, the mice showed no evidence of
cultivable tubercle bacilli (sterile state). But, 12 weeks after INH and PZA treatment was
withdrawn, one-third of the mice developed full-blown active TB, with nearly two-thirds
158 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

displaying disease after 24 weeks. Csillag (1962, 1963, and 1964) considered Mycobacteria as
dimorphic organisms in the same sense as are some pathogenic fungi, for instance,
Histoplasma capsulatum. The usual acid fast form of the mycobacteria was termed ’form I’
and the form which was not acid fast was termed ‘form 2’. When form 2 grown in digest
broth, form 2 strains produced cocci which continued to multiply by binary fission and bud
formation (Csillag, 1964).
These forms were not produced by mycobacteria grown in rich media such as nutrient
broth; Martin’s digest broth, yeast extract and Lab-Lemo beef extract. One year later,
Stewart-Tull (1965) isolated two forms of mycobacteria and mycococci from M. phlei
.Nyka W in 1963, described them as “chromophobic tubercle bacilli” in the lungs of
patients treated by drugs in association with surgery. This organism morphologically
were similar to the acid- fast bacilli, but do not stain with either carbolfuchsin or the
counter stains when applied by the classic Ziehl-Neelsen technique or with any other
aniline dye. In continuation of his work, he submitted the culture of M. tuberculosis,
M. kansasii, and M. phlei to starvation. As a result they lost first their acid fastness, but in
this chromophobic state, they survived for at least 2 years, and after that time, produced
cultures of acid fast bacilli when transferred onto nutrient media. Since these in-vitro
bacilli could recover their original biological properties, it was concluded that those bacilli
in the lung could also become reactivated and cause a relapse of the disease. Some
scientists regard the filterable forms of mycobacteria as being analogous to the so –called
L-forms of the other bacterial genera as they also pass through filters (Thacore and
Willett, 1963). Some other scientists believe that development of the L-form is a mutation
process, while development of the filterable forms is an adaptation of the micro-
organisms to enable them to multiply in unfavorable (Imaeda, 1974; Mattman, 1970:
Ratnam and Chandrasekhar, 1976). In this regards, Takahashi (1979), reported that
tubercle bacilli in caseous lesions seems to be non acid fast, gram negative granules which
may revert into acid fast rods, when the caseous lesion begins to liquefy and form
tuberculous cavity. Similarly, khomenko and colleagues (1987) showed ultra-fine forms of
M. tuberculosis in the walls of open cavities in the lungs of experimental animals by
electron microscopy. These invisible forms of M. tuberculosis are able to revert to the
typical bacterial forms. The initial stage of this process is accompanied by the formation of
coccoid forms of mycobacteria that can be detected when material is inoculated on to
semi-synthetic medium with 10% plasma and by microscopy of the sediment. Lawrence
Wayne (1994) postulated that bacilli recovered from granulomatous lesions had adapted
to a relatively oxygen starved environment so that they would be unable to grow in an
aerated culture and therefore, may be non-cultivable by traditional culture methods
(Wayne and Hayes, 1996). In the Wayne model, cultures of the bacterium are subjected to
gradual self-generated oxygen depletion by incubation in sealed stirred tubes. Upon the
slow shift of aerobic growing M. tuberculosis to anaerobic conditions, the culture is able to
adapt and survive anaerobiosis by shifting down to a state of nonreplicating persistence.
Wayne L showed two phase of growth in mycobacterium under limited oxygen; initially
when the level of drops and the turbidity increased in culture tubes (NRP-1) and in
anaerobic phase when there is no oxygen and no division (NRP-2). Wayne model was a
break through in understanding what may happen to tubercule bacilli in necrotic material
(Wayne and Lin, 1982). Although, Kaprelyants et al (1993) did not consider the bacilli
Morphological Characterization of Mycobacterium tuberculosis 159

obtained by Wayne and Sramek (1994) as dormant because they maintained a high
viability and developed sensitivity to metronidazole when anaerobic, thus indicating
active metabolism. Therefore, from large accumulated data that found in literature, it
become clear that M. tuberculosis can adapt rapidly to changing environment inside and
outside the host (Parrish et al., 1998; Cardona, 2009; Rustad et al., 2009). These capacities
will allow the tubercle bacilli to survive for long time in a dormant state in the lung tissue.
Recently, Peyron et al (2008) developed an in vitro model of human tuberculosis
granulomas. In this model granuloma-specific cell types and their modulation by tubercle
bacilli were characterized. More recently, the complete morphological changes that occurs
in tubercle bacilli under hypoxic conditions viewed under AFM (every 90 days for 48
months) (Velayati et al., 2011). The morphological adaptation classified into two
categories; First was temporary adaptation (from 1 to 18 months of latency) in which cells
undergoing thickening of cell wall (20.5±1.8 nm versus 15.2±1.8 nm, P<0.05), formation of
ovoid cells by “folding phenomena“(65-70%), size reduction (0.8± 0.1 µm versus 2.5±0.5
µm), and budding type of cell division (20-25%) (figure 4).

Fig. 4. Atomic force Microscopy shows M. tuberculosis under 8 months hypoxic condition.
The bacilli becomes round and developed a thickened cell-walls (shows by arrows)

A second feature include changes that accompany development of specialized cells (from 18
to 48 months of latency) i.e., production of spore like cells (0.5 ± 0.2 µm) and their progeny
(filterable non -acid fast forms; 150 to 300 µm in size figure 5). Using AFM, they could
demonstrate that the filterable non-acid fast forms of bacilli are produced from spore –like
cells. These cells were metabolically active and increased their number by symmetrical
typing of division and could be stain by gram staining. Inoculation of these cells could
induce active tuberculosis in mice. Although, it is important to determine how closely the
160 Understanding Tuberculosis – Deciphering the Secret Life of the Bacilli

in vitro models correlate to the state of M. tuberculosis during latent infection. But, if these
models are predictive of human disease, the information they provide in combination with
advances in animal models, imaging and analysis will substantially aid in the development
of drugs capable of killing tubercle bacilli in altered metabolically states, and possibly
shortening the course of TB therapy.

Fig. 5. Atomic force microscopy shows the Latent TB bacilli, after 48 months of latency
(Velayati et al., 2011).

10. Acknowledgments
All the photographs provided here are from personal file and were taken in Microbiology unit” The
Republican Research and Practical Centre for Epidemiology & and Microbiology, Filimonova 23,
Minsk, Belarus”. Thanks are principally due to Prof Gennady Konstantinovich Zhavnerko and prof
Nikolai Nikolaevich Poleschuyk, who help and guide us to take this wonderful pictures from
M. tuberculosis.

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