PNEUMOCOCCUS

A. Morphology
Pneumococci are Gram positive, small (1 µm diameter), slightly elongated
cocci arranged in pairs (diplococci) with the broad ends in apposition. Each
coccus has one end broad or rounded and other pointed (flame shaped or
lanceolate appearance). They are capsulated and the capsule encloses each
pair. The capsule may be demonstrated as a clear halo in India ink
preparation. In old cultures, the capsule is usually lost. They are non-motile
and non-sporing.
B. Culture
Pneumococci have complex nutritional requirement and therefore grow only
in enriched media especially supplemented with blood. They are aerobes and
facultative anaerobes and their growth is improved by 5-10 percent CO2.The
optimum temperature for growth is 37°C(range 25°C to 42°C)and pH 7.8(range
6.5-8.3).
On blood agar, after incubation for 18 hours, the colonies are usually small
(0.51mm),dome shaped, with an area of greenish discolouration (alpha
haemolysis)around them.On prolonged incubation, the colonies become flat, with
raised edges and central umbonation(due to autolysis occurring at centre)which
creates a draughtsman appearance(concentric rings are seen when when viewed
from above). Pneumococci are typically alpha haemolytic but under anaerobic
conditions colonies show beta hamolysis due to liberation of oxygen labile
pneumolysine O by these bacteria.
In liquid medium such as glucose broth, pneumococci produce uniform turbidity.
These cocci readily undergo autolysis in cultures due to the action of intracellular
enzymes. Autolysis is enhanced by bile salts and other surface active agents. This
property is helpful to differentiate it from other streptococci.

C. Pathogenesis
Str. Pneumonia is one of the most common bacteria causing pneumonia, both
lobar and bronchopneumonia. It is also responsible for acute tracheobronchitis
and empyema.
• Lobar Pneumonia
Pneumonia results only when the general resistance is lowered. Common infective
types of Str. pneumoniae include types 1,12 in adults and types 6, 14, 19 and 23
are responsible in children.
• Bronchopneumonia
It is almost always a secondary infection following viral infections of the
respiratory tract. Any serotype of pneumococcus can produce
bronchopneumonia.
Other causative agents responsible for bronchopneumonia include Staph.aureus,
K.pneumoniae, Str.pyogenes.
• Meningitis
It is the most serious of pneumococcal infections. Str.pneumoniae is the second
most important cause of pyogenic meningitis after Nmeningitidis. This disease is
commoner in children. Pneumococcus spreads from the pharynx to the meninges
via bloodstream.
• Other Infections
Pneumococcus may also produce empyema, pericarditis, otitis media, sinusitis,
conjunctivitis, peritonitis and suppurative arthritis, usually as complications of
pneumonia.

MYCOBACTERIUM TUBERCULOSIS
A. Morphology
M. tuberculosis is a slender, straight or slightly curved bacillus with rounded
ends, occurring singly, in pairs or in small clumps. It measures, 1-4 µm x 0.2 - 0.8
µm (average 3 µm x 0.3 µm) in size. These bacilli are acid-fast, non-sporing, non-
capsulated and non-motile. Ziehl Neelsen staining is useful to study the
morphology of these organisms. With this stain, tubercle bacilli are seen bright
red (acid-fast), while the tissue cells and other organisms are stained blue
.Tubercle bacilli may also be stained with the fluorescent dyes (aurarnine 0 ,
rhodarnine) and appear yellow luminous bacilli under the fluorescent
microscope. Beaded or barred forms are frequently seen in M tuberculosis. They
are Gram positive but are difficult to stain with the Gram stain due to the failure
of the dye to penetrate the cell wall.

B. Culture
M. tuberculosis is an obligate aerobe whereas M. bovis is microaerophilic on
primary isolation, becoming aerobic on subculture. The bacilli grow slowly
(generation time 14-15 hours) and colonies appear only in about two weeks and
sometimes it may take up to 6-8 weeks. Optimum temperature for growth is 37°C
(range 3-0°C). Optimum pH is 6.4 to 7.0. Tubercle bacilli can grow on a wide range
of enriched culture media but Lowenstein-Jensen (LJ) medium is most commonly
used. This medium consists of beaten eggs, asparagine, mineral salts, malachite
green and glycerol or sodium pyruvate.It is solidified by heating (inspissation). It
is one of the media which are solid without incorporation of agar. In this medium
egg acts as a solidifying agent. Malachite green inhibits the growth of organisms
other than mycobacteria and provides a colour to the medium. The addition of
glycerol improves the growth of human type of M tuberculosis, while it is without
any effect or even inhibitory to M. bovis. Sodium pyruvate improves the growth
of both M. tuberculosis and M. bovis. Colonies of M. tuberculosis are dry, rough,
buff coloured, raised, with a wrinkled surface.They are tenacious and not easily
emulsified. M. tuberculosis has a luxuriant growth (eugonic growth) in culture as
compared to sparsely grown (dysgonic growth) M. bovis. In liquid media, the
bacilli grow as surface pellicle due to hydrophobic properties of their cell wall.
Liquid media are generally used for sensitivity testing, preparation of antigens
and vaccines. In automated culture methods, liquid media are used for growing
mycobacteria. Various culture media used for mycobacterial isolation include
Lowenstein-Jensen (LJ) medium.
C. Pathogenesis
The infection is commonly acquired by inhalation of infected droplets coughed or
sneezed into the air by a patient with pulmonary tuberculosis. Tubercle bacilli
are engulfed by macrophages but they survive and multiply in macrophages.
These lyse the host cell, infect other macrophages and sometimes disseminate to
other parts of lung and elsewhere in the body. The cell mediated immunity (CMI)
plays a major role to interact with these macrophages whereas humoral
immunity appears to be irrelevant, CD4+ helper T cells secrete interferon
gamma, interleukin 2, tumour necrosis factor a and others exerting different
biological effects. It may result in protective immunity or delayed type
hypersensitivity (DTH) reaction. Th-1 dependent cytokines activate macrophages
to kill intracellular mycobacteria and thus result in protective immunity. Th-2
cytokines induce delayed type hypersensitivity (DTH), tissue destruction and
progressive disease. Human tuberculosis is divisible into primary and secondary
forms.
• Primary Tuberculosis
Inhaled tubercle bacilli are engulfed by alveolar macrophages in which they
replicate to form a lesion called Ghan focus. It is frequently found in the lower
lobe or lower part of the upper lobe. Some bacilli are transported to the hilar
lymph nodes. The Ghon focus together with the enlarged hilar lymph nodes is
called the primary complex.
• Secondary (Post Primary) Tuberculosis
It is caused by reactivation of the primary lesion (endogenous) or by exogenous
reinfection. Reactivation tuberculosis is likely to occur in immunocompromised
individuals. Granulomas of secondary tuberculosis most often occur in the
apex of lungs. The necrotic element of the reaction causes tissue destruction
and the formation of large area of caseation termed tuberculomas. Presence of
caseous necrosis and cavities are two special features of secondary
tuberculosis. Cavities may rupture into blood vessels, spreading mycobacteria
throughout the body, and break into airways, releasing the organisms in
aerosols and sputum (open tuberculosis).