Assignment Topic

“CLASSIFICATION OF BACTERIA”

MICROBIOLOGY

Submitted To: Dr. FARKHANDA JABEEN

Submitted By: SHAFIQ AHMED

Roll No. M16-39

M.Sc 1st Semester

Department of Botany

University of the Punjab

Lahore-Pakistan
Outline:

o Non photosynthetic gliding Bacteria

o The Myxobacteria
o Gram positive rods
o Endospore forming rods and cocci
o Regular nonsporing gram positive rods
o Irregular nonsporing gram positive rods
o The Mycobacteria
Non photosynthetic gliding Bacteria:

Gliding bacterium,  plurals Gliding Bacteria, any member of a heterogeneous group of

microorganisms that exhibit creeping or gliding forms of movement on solid substrata.
Gliding bacteria are generally gram-negative and do not possess flagella. The complex
mechanisms by which they move have not been fully ascertained, and such mechanisms differ
among various species. The gliding bacteria include the fruiting myxobacteria
(e.g., Myxococcus and Cystobacter); the cytophaga group of nonfruiting, nonphotosynthetic rods
or chains (e.g., Cytophaga and Sporocytophaga); and certain filamentous bacteria such
as Thiothrix, Simonsiella, and Leucothrix. Gliding bacteria are found in soils and in fresh and
marine waters. Simonsiella inhabits the oral cavities of humans and animals. The filaments
of Beggiatoa resemble filamentous cyanobacteria; they are found in sulfur-rich deposits and
hydrothermal vents.

Myxobacteria:

The myxobacteria ("slime bacteria") are a group of bacteria that predominantly live in the

soil and feed on insoluble organic substances. The myxobacteria have very large genomes,
relative to other bacteria, e.g. 9–10 million nucleotides. Sorangium cellulosum has the largest
bacterial genome, at 13.0 million nucleotides. Myxobacteria are included among the delta
group of proteobacteria, a large taxon of Gram-negative forms.
Myxobacteria can move actively by gliding. They typically travel in swarms (also known as wolf
packs), containing many cells kept together by intercellular molecular signals. Individuals
benefit from aggregation as it allows accumulation of the extracellular enzymes that are used to
digest food; this in turn increases feeding efficiency. Myxobacteria produce a number of
biomedically and industrially useful chemicals, such as antibiotics, and export those chemicals
outside the cell.

Life cycle:
When nutrients are scarce, myxobacterial cells aggregate into fruiting bodies (not to be
confused with those in fungi), a process long-thought to be mediated by chemotaxis but now
considered to be a function of a form of contact-mediated signaling.[3][4] These fruiting bodies can
take different shapes and colors, depending on the species. Within the fruiting bodies, cells begin
as rod-shaped vegetative cells, and develop into rounded myxospores with thick cell walls.
These myxospores, analogous to spores in other organisms, are more likely to survive until
nutrients are more plentiful. The fruiting process is thought to benefit myxobacteria by ensuring
that cell growth is resumed with a group (swarm) of myxobacteria, rather than as isolated cells.
Similar life cycles have developed among certain amoebae, called cellular slime molds.

At a molecular level, initiation of fruiting body development is regulated by Pxr sRNA.[5][6]

Myxobacteria such as Myxococcus Xanthus and Stigmatella aurantiaca are used as model

organisms for the study of development

Clinical use:
Metabolites secreted by Sorangium cellulosum known as epothilones have been noted to
have antineoplastic activity. This has led to the development of analogs which mimic its activity.
One such analog, known as Ixabepilone is a U.S. Food and Drug Administration approved
chemotherapy agent for the treatment of metastatic breast cancer.[7]

Myxobacteria are also known to produce Gephyronic acid, an inhibitor of eukaryotic protein

synthesis and a potential agent for cancer chemotherapy.
Gram Positive Rods:

Gram-positive bacteria are bacteria that give a positive result in the Gram stain test. Gram-

positive bacteria take up the crystal violet stain used in the test, and then appear to be purple
colored when seen through a microscope. This is because the thick peptidoglycan layer in the
bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the de
colorization stage of the test.

Gram-negative bacteria cannot retain the violet stain after the de colorization step; alcohol used in
this stage degrades the outer membrane of gram-negative cells making the cell wall more porous
and incapable of retaining the crystal violet stain. Their peptidoglycan layer is much thinner and
sandwiched between an inner cell membrane and a bacterial outer membrane, causing them to take
up the counter stain (safranin or fuchsine) and appear red or pink.

Despite their thicker peptidoglycan layer, gram-positive bacteria are more receptive
to antibiotics than gram-negative, due to the absence of the outer membrane.
Gram-positive bacilli belong to a class of rod-shaped bacteria that acquire a violet color
when subjected to the Gram staining method. Aside from its characteristic shape, this
class of bacteria has a thick peptidoglycan layer, which lends itself to better absorption
of antibiotics.
Many Gram-positive bacilli have caused numerous diseases around the world.
Mycobacterium tuberculosis causes severe lung disorders and is transmitted
through the air by sneezing or coughing. Listeria monocytogenes is a facultative
bacteria species that causes listeriosis, one of the primary causes of meningitis and
meningoencephalitis. Mycobacterium leprae infection in humans results in Hansen's
disease, more commonly known as leprosy. Bacillus anthracis is the main agent of
anthrax, the fatal disease considered a bioterrorism threat in 2001 when bacterial
spores were delivered in regular mail.

In contrast, some Gram-positive bacilli are safe for humans and are even infused in
food. Bacillus subtilis is a spore-forming bacteria used in manufacture of the
Japanese food natto and is also used in antibiotics to treat urinary tract infections.

Gram staining is a bacterial differentiation method that categorizes various species

of bacteria according to their response to a violet stain. This procedure relies on the
absorption of a crystal violet dye by the bacteria's peptidoglycan layer, a layer
present only in Gram-positive bacteria.
Endospore Forming gram positive rods:
Clostridium botulinum—anaerobic—adult and infant botulism

Clostridium perfringens—anaerobic—diarrhea

Bacillus cereus—facultatively anaerobic—gastroenteritis (diarrhea and vomiting)

Why do bacteria form endospores.

• Survival of the bacterial species

 • Endospores carry the genomic information of the vegetative cell

Vegetative cells vs. endospores

• Vegetative cells are metabolically active

(taking in nutrients, converting nutrients into energy and biomass, expelling

wastes, growing and dividing)

Endospores are dormant—metabolically inactive (but contains the genetic material of the
vegetative cell).

Why are endospores dormant?

• Filled with SASPS (small acid soluble proteins that protect DNA)

• Has low water activity

• Has relatively few metabolic enzymes

• Surrounded by a tough keratin-like coat.

How does being dormant benefit an endospore:

• Can survive the following conditions

drying

high heat

UV irradiation

low pH

high osmolarity

low temperature

Conditions that most vegetative cells cannot survive

Life cycle of an endospore

• Vegetative cell (unpleasant conditions)
 • Veg. cell replicates DNA, forms a septum 1/3 of the length of the cell and pumps DNA
into the region

• Endospore develops at that site and is finally surrounded by a tough keratin like coat

• Veg. cell dies releasing the mature endospore

• Free mature endospore (pleasant conditions)

• Endospore germinates to become a vegetative cell.

Clostridium botulinum:
• Causes botulism (food posioning)

• Causes infant botulism from honey

• Abundant in soil throughout the world

Type A C. botulinum—found in neutral/alkaline soil west of the Mississippi River

Type B C. botulinum—found in eastern part of the country

Type C C. botulinum—found in wet soils (can effect fish)

Botulism cases are rare

• Foodborn botulism—50 cases per annum usually due to home canning of food (Type A
and Type B) or preserved fish (Type E)

• Infant botulism—100 cases per annum usually due to spores present in honey that is
introduced to formula.

Foodborn botulism—toxin formed in food

• Canning—endospores are not eradicated if the temperature is not sufficiently high

• Spores are present in a nutrient rich environment that is also anaerobic

• The spores germinate and become vegetative bacteria

 • The vegetative bacteria produce botulism toxin—a potent neurotoxin

Infant botulism—(3-20 weeks old)

• Toxin is formed in the infant’s colon

• Honey has a high osmolarity; therefore, the endospores cannot germinate

• Honey is mixed with infant formula that dilutes the honey—the infant drinks the solution

• The spores are introduced into the stomach and move to the colon

• The spores germinate in the colon

• The vegetative cells produce botulism neurotoxin in the colon

Clinical diseases caused by botulism neurotoxin (cause)

• Foodborn botulism--12-36 hours after ingestion toxin travels to different parts of the
body

• Botulism toxin binds to acetocholine receptors on neural cells

• Impulses are not sent from one neural cell to another

• Muscles (smooth, involutary, motor) are not stimulated by the neurons and cannot move

• This causes flaccid paralysis of the muscles

Clinical diseases caused by botulism neurotoxin (effect)

• Flaccid paralysis of muscles

• Cranial nerves are the first affected

Double vision/blurred vision

Difficulty swallowing

• Muscles in the arms and legs are next to be affected

• Diaphragm muscles are affected causing difficulty in breathing

• There is no fever or cell death

 • END RESULT:: paralysis and respiratory failure.

Treatment:
• Deliver antitoxin to the three known botulism neurotoxin

• The antitoxin binds to the free circulating neurotoxin and prevents it from binding to
acetocholine receptors

• N.B. Bound neurotoxin cannot be inactivated as it is already bound to acetocholine

receptors

• Supportive therapy—intubation until neurons regenerates.

Infant botulism-diseases and treatment

• Disease is slow in occurring because toxin cannot be readily absorbed from the colon

• 2-3 days constipation

• Difficulty swallowing—poor suckling response

• Flaccid baby

• Hospitalization and intubation may be required

• Prognosis is usually good so no antitoxin is required

• N.B. SIDS has been attributed in part to infant botulism.

• Irregular nonsporing gram positive rods:

• Gram-Positive Irregular Non- Spore-Forming Bacilli
• • Pleomorphic and stain unevenly • 20 genera _ Corynobacterium _ Mycobacterium _
Nocardia
• CORYNEBACTERIA (AEROBES)
• - Causes localized inflammation (pseudomembrane, greyish white exudate ) and
generalized toxaemia
• - Prevalent in baby’s after 3-6 months (that’s why DPT is given at 3, 5, 7 months,
boosters at 18 months and at school entry), very high in young children
• Morphology
• • Gram/+ve/palisade/ Chineseletter arrangement
• • Irregular swellings at one end -club shaped.
• • Corynebacteria tend to pleomorphism in microscopic and colonial morphology.
• • On blood agar Small granular & gray with irregular edges and may have small zones of
hemolysis.
• • Grow aerobically on ordinary media
• a. Corynobacterium diphtheriae Normal flora of nasopharynx in about 10% –
Diphtheria caused when infected by lysogenicbacteriophage
• b. Diptheroids – Normal flora of skin – Usual contaminants of samples – Can cause
disease in ‘compromised’host
• C. ulcerans C. haemolyticum C.Ps.diphtericum C.Xerosis
• • Rare in developed countries/ third world countries
• • Nose, Nasopharynx, skin aerobic, facultatively anaerobic
• • Nasal carriers are very dangerous
• Epidemiology
• • It is rare in developing countries, a disease of the third world countries. Still highly
prevalent in the former Soviet Union.
• • Spread through droplets.
• Corynebacterium diphtheriae
• • 2 – Transmission • Close contact with the droplets from human carriers or active
infections • Occasionally fomites or contaminated milk
• • Loeffler's serum slope Blood telurite agar (black colonies)
• • Morphological differences
• • Three biotypes Gravis (severe) Inter-medius (intermediate) Mitis (mild)
• Types of Diphtheria
• • Faucial • Laryngeal • Nasal • Conjunctival • Vulvovaginal • Otitic • Cutaneous around
the mouth and the nose
• Effect of toxins 1. Local(inflammatory reaction, low grade fever,nausea, vomitting,
enlarged cervical nodes, sever swelling in the neck) 2. General Toxaemia and acts on the
myocardium and on motor nerves and adrenals Complications a, pseudomembrane may
 extend to larynx and cause airway obstruction b.myocarditis /Polyneuropathy •
Degenerative changes in the liver adrenals, kidney's
• Pathology
• •Toxin is absorbed in the mucus membrane and causes destruction of epethelium and
causes a superficial inflammatory respons.
• •Necrotic epethelium becomes embeded in exuding fibrin and red and white cells, with
bacteria-
• •Grayish pseudomembrane is formed over the tonsilas and pharynx and larynx.
• • How to identify the immune persons Shick test – suitably diluted stabilized toxin
intradermally(0.2ml), localized erythema (1-3cm) in 2-4 days, means no or little
antibodies 0.005U/ml
• Corynebacterium diphtheriae
• • 4 – Factors of pathogenicity • Non invasive bacteria • Local multiplication (mucus) •
Secretion of diphtherotoxin – Local lesions – diffusion
• Corynebacterium diphtheriae
• • 4 – Factors of pathogenicity • Proteic toxin (cytotoxin) – fragment B binds to and
endocytosed by mammalian target cells in the heart & nervous system – fragment A
inhibit protein synthesis of the cell • antigenicity – Protective antibodies – vaccination
(toxine formaline anatoxine)
• Pseudomembrane

• Diagnosis • Direct smear - Albert's stain • Culture - Loffler's serum slope/blood

agar/blood telurite agar
• Check the toxigenicity • Animal inoculation Death within 96 hrs Guinea pigs/rabbits –
Elek’s plate test – PCR
• Elek’s test
• Elek's plate test Filter paper with antitoxin
• Precipitation
• Strain
• Management – 1. Patients - isolation of the patient / bed rest/antibiotic
treatment/antitoxins (horse serum)DAT 10000-20000U ,IV
• Penicillin/erythromycin/teracycline/rifam picin/clindamycin 2. Contacts – immunize if
not (toxoid) – adults should be shick tested or given low dose as immunization of
immune adults can result in severe reaction. - prophylactic antibiotic – erythromycin -
swab nose and throats of contacts
• Corynebacterium diphtheriae
• • 6 – Management: - Prevalent in baby’s after 3-6 months (that’s why DPaT is given at 3,
5, 7 months, boosters at 18 months and at school entry), very high in young children -
Older children and adults Td
• Gaston Ramon
• 3. Community – immunization
• DIPHTHERIA
• DIAGNOSIS
• Clinical suspicion Swab for culture Toxin production
• TREATMENT
• Penicillin Anti-diphthereticserum Maintaining airway Supportive
• PREVENTION
• Immunization (toxoid)
• Propionibacterium
• • Similar to corynobacterium • Anaerobic, nontoxigenic • Propionibacterium acne •
Resident of pilosebaceous glands of human skin and URT • Lipase production • Acne
vulgaris
• ACTINOMYCETES (FACULTATIVELY ANAEROBES)
• •Fermentative gp: Actinomyces, Arcanobacterium and Rothia
• •Oxidative gp : Actinomadura (actinomycetoma), Nocardia (nocardiosis), Streptomyces
and related species.
• Actinomycosis
• • A. israelii – the commonest • A .meyeri • A.naeslundii • A.odontolyticus • A. viscosus
• 6. Actinomyces israelii
• • Has branching filaments • Facultative anaerobes • Normal flora of oral cavity, tonsils
and intestine • Causes ‘Actinomycosis’ characterised by multiple abscess and granuloma
formation • Tissue destruction, fibrosis and sinus formation
• ACTINOMYCOSIS
• • Mostly in cervico-facial region • Endogenous infection • Can get – Thoracic
actinomycosis (aspiration) – Pelvic actinomycosis (IUCD) – Rarely haematogenous
spread • Treatment – Surgical – Long term penicillin
• Cervicofacial disease
• Diagnosis
• • Specimens – open biopsy, aspiration material
• • Sulphur granules (yellowish myecelial masses)
• • The discharge should mix with sterile saline in a universal bottle and allow to stand,
particles will separate out.
• • Place between 2 slides
• • Crush and gram stain
• • Gram positive branching filaments
• ACTINOMYCOSIS
• Nocardiosis
• • N.brasiliensis :pulmonary pathogen • N.asteroides and N.caviae : opportunists •
Infections: - Pulmonary - Cutaneous - Subcutaneous
• Nocardiosis
• • Branched, strictly aerobic bacillus • Environmental saprophytes (exogenous infection) •
Lightly acid-fast • Uncommon causes of opportunistic pulmonary disease • Causes
primary post-traumatic or post- inoculation lung disease
• Cutaneous nocardiasis
•
• Nocardiosis
• • Diagnosis and treatment: sputum, pus, CSF, biopsy gram positive coccobacilli with
braches Cotrimoxazol, Amikacin, Imepenem, Cefotaxim
Mycobacterium tuberculosis Morphology and identification

A. Typical organisms: In tissue, tubercle bacilli are thin straight rods with variable morphology from
one species to another.  Mycobacteria cannot be classified as gram +ve or -ve.  They are
characterized by 'acid-fastness i.e. 'acid-alcohol' quickly decolorizes all bacteria except the
mycobacteria, acid- fastness depends on the integrity of the waxy envelope .The Ziehl-Neelsen
technique of staining is employed for identification of acid-fast bacteria.

B. Culture:

There are three general formulations that can be used for both the nonselective and the selective
media:-

1) Semi-synthetic agar media (eg, Middlebrook 7H10 and 7H11) contain salts, vitamins cofactors, oleic
acid, albumin, catalase, glycerol, glucose, and malachite green. Large inocula yield growth on these
media in several weeks. These media may be less sensitive than other media for primary isolation of
mycobacteria.

2) Inspissated egg media (eg, Lowenstein-Jensen) contain salts, glycerol, and complex organic
substances (eg, fresh eggs, egg yolks, potato flour, and other ingredients).Malachite green is included to
inhibit other bacteria. Small inocula in specimens from patients will grow on these media in 3-6 weeks.
These media with added antibiotics are used as selective media.

3) Broth media: broth media (eg, Middlebrook 7H9 and 7H12) support the proliferation of small inocula.
Mycobacteria grow in clumps or masses because of the hydrophobic character of the cell surface, and
added antibiotics.

C. Growth characteristics: 1) Mycobacteria are obligate aerobes.

2) Increased Co2 tension enhances growth. 3) Biochemical activities are not characteristics, and the
growth rate is much slower than that of most bacteria.

4) Saprophytic forms tend to grow more rapidly, to proliferate well at 22-33 cC. To produce more
pigment, and to be less acid-fast than pathogenic forms.

D. Reaction to physical and chemical agent:  Mycobacteria tend to be more resistant to chemical
agents than other bacteria because of the hydrophobic nature of the cell surface and their clumped
growth. Dyes (malachite green)or antibacterial agents (eg, penicillin)that are bacteriostatic to other
bacteria can be incorporated into media without inhibiting the growth of tubercle bacilli. Acids and
alkalies permit the survival of some tubercle bacilli and are used to help eliminate contaminating
organisms and for 'concentration' of clinical specimens.  Tubercle bacilli are resistant to drying and
survive for long periods in dried sputum. E. Variation. F. Pathogenicity of Mycobacteria:  Humans and
guinea pigs are highly susceptible to M.tuberculosis infection, whereas fowl and cattle are resistant 

M.tuberculosis and M bovis are equally pathogenic for humans. The route of infection
(respiratory versus intestinal determines the pattern of lesions.  Some 'atypical' mycobacteria(eg,
Mycobacterium kansasii ) produce human disease indistinguishable from tuberculosis, other (eg,
M.fortuitum ) cause only surface lesion and or act as opportunists.

Constituents of tubercle Bacilli

The constituents listed below are found mainly in cell walls. Mycobacterial cell walls can induce
delayed hypersensitivity and some resistance to infection and can replace whole mycobacterial cells in
Freund's adjuvant.

A. Lipid Mycobacteria are rich in lipids. These include mycolic acids, waxes, and phosphatides. The lipids
are largely bound to proteins and polysaccharides. Lipids are to some extent responsible for acid
fastness.

Virulent strains of tubercle bacilli form microscopic "serpentine cords" in which acid-fast bacilli
are arranged in parallel chains. Cord formation is correlated with virulence. A "cord factor" inhibits
migration of leukocytes, causes chronic granulomas, and can serve as an immunologic "adjuvant".

B. Proteins: They elicit the tuberculin reaction. They can also elicit the formation of variety of
antibodies.

C. Polysaccharides: They can induce the immediate type of hypersensitivity and can serve as antigens in
reactions with sera of infected persons.

Pathogenesis
 Mycobacteria in droplets are inhaled and reach the alveoli. The disease results from
establishment and proliferation of virulent organisms and interactions with the host. Resistance and
hypersensitivity of the host greatly influence the development of the disease.

Pathology

The production and development of lesions and their healing or progression are determined chiefly by:

1- The number of mycobacteria in the inoculum and their subsequent multiplication.

2- The resistance and hypersensitivity of the host.

A. Two principal lesions:

1. Exudative type  this consists of an acute inflammatory reaction, with edema fluid,
polymorphonuclear leukocytes, and, later, monocytes around the tubercle bacilli. This type is seen
particularly in lung tissue. It may heal; it may lead to massive necrosis of tissue; or it may develop into
the second (productive) type of lesion. During the exudative phase, the tuberculin test becomes
positive. 2. Productive type  when fully developed it consists of three zones:
(1) a central area of large, giant cells containing tubercle bacilli;
(2) a mid-zone of pale epithelioid cells;

(3) a peripheral zone of fibroblasts, lymphocytes, and monocytes. Later the central area undergoes
caseation necrosis. Such a lesion is called a tubercle. It may break into a bronchus, empty its contents
there, and form a cavity. It may subsequently heal by fibrosis or calcification.

B. Spread of Organisms in the Host:

By direct extension through the lymphatic channels and bloodstream, and via the bronchi and
gastrointestinal tract. The bloodstream distributes bacilli to all organs (miliary distribution). If a
caseating lesion discharges its contents into a bronchus, they are aspirated and distributed to other
parts of the lungs or are swallowed and passed into the stomach and intestines.

C. Intracellular Sites of Growth:

 Mycobacteria establish themselves in tissue; intracellularly in monocytes, reticuloendothelial
cells, and giant cells. That makes chemotherapy difficult and favors microbial persistence. Within the
cells of immune animals, multiplication of tubercle bacilli is greatly inhibited.

PRIMARY INFECTION & REACTION

When a host has first contact with tubercle bacilli, the following features are observed: a. An
acute exudative lesion develops and rapidly spreads to the lymphatics and regional lymph nodes. b. The
lymph node undergoes massive caseation. c. The tuberculin test becomes positive.

in primary infection, the involvement may be in any part of the lung but is most often at the base.

The reactivation type is caused by tubercle bacilli that have survived the primary lesion. Reactivation
tuberculosis is characterized by

a. Chronic tissue lesions.

b. Regional lymph nodes are only slightly involved.(Ghon complex).

The reactivation type almost always begins at the apex of the lung where oxygen tension (Po2) is
highest.

These differences between primary infection and re infection or reactivation are attributed to:

1) Resistance
2) hypersensitivity induced by the first infection of the host with tubercle bacilli "Koch's phenomena".

Immunity and Hypersensitivity

1) Development of cellular immunity during the initial infection. 2) Antibodies form against a
variety of the cellular constituents of the tubercle bacilli 3) In the course of primary infection, the host
develops hypersensitivity to the tubercle bacilli. 4) This is made evident by the development of a
positive tuberculin reaction.

Tuberculin Test
A-Material:

Old tuberculin is a concentrated filtrate of broth in which tubercle bacilli have grown for 6 weeks.
A Purified protein derivative ( P P D ) is obtained  It's standardized by TU "Tuberculin Units".

Doses 5T U, 250TU according to strength required.

B. Reactions to Tuberculin:

In an individual who has not had contact with mycobacteria, there's no reaction.  An individual
who has had a primary infection with tubercle bacilli develops induration, edema, and erythema in 24-
48 hours. The skin test should be read 48 or 72 hours. Induration 10mm or more in diameter.positive
tests tend to persist for several days.  The tuberculin test becomes positive within 4-6 weeks after
infection.  It may be negative in the presence of tuberculous infection when "anergy" develops due to:

1) Overwhelming tuberculosis

2) Measles 2) Hodgkin’s disease

3) Sarcoidosis

4) AIDS

5) Immuno suppression. After BCG vaccination, a positive test may last for only3-7 years.

C. Interpretation of Tuberculin test

A positive tuberculin test indicates that an individual has been infected in the past or continue
to carry viable mycobacteria in some tissues. It does not imply that active disease or immunity to
disease is present .tuberculin- positive persons are at risk of developing disease from reactivation of the
primary infection.

Diagnostic laboratory tests

A positive tuberculin test does not prove the presence of active disease due to tubercle bacilli.
Isolation of tubercle bacilli provides such proof:
A. Specimens consist of fresh sputum, gastric washings, urine, pleural fluid, C5F, joint fluid, biopsy
material, blood, or other suspected material.

B. Decontamination and Concentration of Specimens:

Specimens from sputum with NaOH, neutralized with buffer, and concentrated by centrifugation  Used
for acid-fast stains and for culture.

C. Smears: • Examined for acid-fast bacilli by Ziehl-Neelsen staining.

Fluorescence microscopy with auramine-rhodamine stain is more sensitive than acid-fast stain.

D. Culture, Identification, and Susceptibility Testing:

A selective agar media (e.g. Lowenstein-Jensen or middlebrook 7H10/7H11).Incubation is at

37°C in 5-10% C02 for up to 8 weeks.  It is medically important to characterize and separate M.
tuberculosis from all other species of mycobacteria.  Conventional methods for identification of
mycobacteria include observation of rate of growth, colony morphology, pigmentation, and biochemical
profiles. Growth rate separates the rapid growers <7 days, from other mycobacteria.  Molecular
probes provide a rapid, sensitive, and specific method for identification of mycobacteria.

E. Antigen Detection, serology and anti-gene detection (PCR)

The polymerase chain reaction holds great promise for the rapid and direct detection of M.
tuberculosis in clinical specimens- the PCR test is approved for this use.

Prevention & Control:

1-Prompt and effective treatment of patients with active tuberculosis

2-Drug treatment of asymptomatic tuberculin-positive persons (e.g. children)-receive

immunosuppressive drugs.

3- Nonspecific factors may reduce host resistance include starvation, gastrectomy, and suppression of
cellular immunity by drugs.
4- Immunization Various living a virulent tubercle bacilli, particularly BCG (Bacillus Calmette-Guerinan
attenuated bovine organism). Vaccination is a substitute for primary infection with virulent tubercle
bacilli without the danger inherent in the latter given to children.

5- The eradication of tuberculosis in cattle and the pasteurization of milk have greatly reduced M bovis
infections.