Download BSc/Diploma in Medical Laboratory Technology 3 BLT302

PROGRAM
BSc/Diploma in Medical Laboratory Technology
SEMESTER
3
SUBJECT
BLT302 - MICROBIOLOGY-II: BACTERIOLOGY AND VIROLOGY
BOOK ID
B1826
SESSION
Winter 2015
No
Q 1
Question/Answer key
Marks Total Marks
10
Describe the morphology, cultural characteristics, biochemical reactions,
pathogenesis, clinical manifestation and laboratory diagnosis of Mycobacterium
tuberculosis.
( Unit 3 ; Section 3.2 )
A 1
Morphology, cultural characteristics, biochemical reactions, pathogenesis,
clinical manifestation and laboratory diagnosis
• Morphology
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• Tubercle bacilli are thin straight rods that measure about 0.4 × 3 μm in size on
media and appear as coccoid and filamentous forms.
• Mycobacteria cannot be classified as either Gram-positive or Gram-negative.
The Ziehl-Neelsen (ZN) technique of staining or acid fast staining is employed for
identification of acid-fast bacteria that are also demonstrated by yellow-orange
fluorescence in auramine and rhodamine staining methods.
• Cultural Characteristics
Three types of media formulations can be used as both the nonselective and
selective media as follows:
• 1. Semi-synthetic Agar Media
• (a) Middlebrook 7H10 and 7H1: It contains defined salts, vitamins, cofactors,
oleic acid, albumin, catalase, glycerol, glucose, and malachite green.
• (b) 7H11 medium: It contains casein hydrolysate. Both these media are used
for observing colony morphology, for susceptibility testing.
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• 2. Inspissated egg-based media
Löwenstein-Jensen medium which contains salts, glycerol, and complex organic
substances (e.g. fresh eggs or egg yolks, potato flour and Malachite green) is
used to inhibit other bacteria.
• 3. Broth Media
Middlebrook 7H9 and 7H12 are examples of broth media where the growth is
more rapid than on other media.
• Mycobacteria are obligate aerobes. Their growth rate is much slower than
most bacteria. The doubling time is about 18 hours. The bacilli that are
saprophytic grow more rapidly. They proliferate at 22–33°C to appear more
pigmented and less acid-fast than pathogenic forms.
• Biochemical Reactions
• The biochemical reactions shown by mycobacteria are as follows:
• Niacin test: M. tuberculosis (positive)
• Aryl sulphatase test: Positive by atypical mycobacteria only
• Neutral red test: Virulent mycobactria are positive
• Nitrate test: M. tuberculosis (positive)
• Catalase test: M. tuberculosis (weakly positive); atypical mycobacteria
(strongly positive)
• Peroxidase test: M. tuberculosis (positive); atypical mycobacteria (negative)
• Pathogenesis and Clinical Manifestations
• It is possible for you to determine the route of infection, whether it is respiratory
or intestinal, by observing the pattern of lesion. Mycobacteria present in droplets
are inhaled to reach alveoli where there is establishment and proliferation of
virulent organisms and interactions with the host. The resistance and
hypersensitivity of the host influences the development of the disease. The two
types of lesions caused by M. tuberculosis are as follows:
• Exudative type: An acute inflammatory reaction, with oedema, fluid,
polymorphonuclear leukocytes, and, later, monocytes around the tubercle
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bacilli.
• Productive type: A chronic granuloma develops, followed by peripheral fibrous
tissue development. A caseous tubercle breaks into a bronchus. It then pours its
contents and forms a cavity that may heal by fibrosis or get calcified.
• Antibodies are formed to cellular constituents of the tubercle bacilli that are
determined by serologic tests though none of these serologic reactions bear any
unequivocal relation to the immune state of the host. Hypersensitivity and
resistance are distinct aspects of related cell-mediated reactions.
• Laboratory Diagnosis
• Laboratory diagnosis involves the culturing of M. tuberculosis. The specimen is
collected from the patient. The various laboratory diagnosis methods are as
follows:
• 1. Sputum: Sputum smears and cultures are done by fluorescence microscopy
(auramine-rhodamine staining) for sputum sample. This technique is more
sensitive than conventional Ziehl-Neelsen staining.
• 2. Other sampling: In case a patient is unable to give a sputum sample, gastric
washings, laryngeal swab, bronchoscopy (with bronchoalveolar lavage, bronchial
washings, and/or transbronchial biopsy) and fine needle aspiration (transtracheal
or transbronchial) are employed.
• 3. PCR: PCR or gene probe tests are done in case a smear is positive in order
to distinguish
M. tuberculosis from other mycobacteria.atient. The various laboratory diagnosis
methods are as follows:
• 4. Routine culture employs Löwenstein-Jensen (LJ), Kirchner, or Middlebrook
media (7H9, 7H10, and 7H11).
• 5. Automated systems are faster by the MB/BacT, BACTEC 9000,
VersaTREK, and the Mycobacterial Growth Indicator Tube (MGIT).
• 6. ALS Assay: Antibody in Lymphocyte Supernatant (ALS) Assay is based on
the antibodies present in blood circulation of a patient for a short period of time in
response to TB-antigens during active TB infection.
• 7. Tuberculin skin test is done by the Mantoux skin test
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• 8. Adenosine deaminase test: It is used in pleural fluid samples for diagnosis of
pleural TB, where sensitivity is very high like TB meningitis.
• 9. Nucleic acid amplification tests (NAAT) are also used which include
polymerase chain reaction (PCR) technique or Transcription mediated
amplification (TMA).
• Tuberculin Test
You can obtain purified protein derivative (PPD) by chemical fractionation of old
tuberculin. It is standardized in terms of tuberculin units (TU) and is injected into a
hypersensitive host. An individual who has had a primary infection with tubercle
bacilli develops induration, oedema, and erythema within 24–48 hours. In case of
intense reactions, central necrosis is observed. The skin test is read in 48 or 72
hours and is considered positive if 5 TU give an induration 10 mm or more. It may
be negative in the presence of tuberculous infection when ‘anergy’ (a state of
immune unresponsiveness) develops due to tuberculosis, measles, Hodgkin’s
disease, sarcoidosis, AIDS, or immune suppression. Persons that were
PPDpositive
years earlier and are healthy now may fail to give a positive skin test. A positive
tuberculin test indicates that an individual has been infected in the past and
continues to carry viable mycobacteria in some tissue.
Q 2
10
Discuss the various routes of transmission of infection.
( Unit 1 ; Section 1.3 )
A 2
Various routes of transmission of infection
• The term ‘transmission’ means transfer of microorganisms directly from the
diseased person to another person, which occurs by the following means:
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• Droplet contact during coughing or sneezing on another person
• Direct physical contact with the infected person (touching an infected person or
through sexual contact)
• Indirect physical contact which is usually by touching contaminated soil or a
contaminated surface
• Through air, but it can only happen when the microorganism can sustain in the
air for a long time
• Through faecal–oral route (through contaminated food or water sources)
• Transmission can also occur indirectly through other organisms. This can
either be a vector (like a mosquito) or an intermediate host (like tapeworm in pigs
which can be transmitted to humans ingesting half-cooked pork).
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• Transmission of disease takes place either vertically or horizontally.
• Horizontal disease transmission: In this form, transmission takes place within
the same generation. Horizontal transmission can occur by either direct contact
through licking, touching and biting, or by indirect contact such as, by coughing or
sneezing.
• Vertical disease transmission: In this form, a disease-causing agent is passed
vertically from the parent to the offspring, i.e. it occurs between generations (e.g.
perinatal transmission).
• The different routes for transmission of infection are following:
• (i) Droplet Contact:
It is also known as the respiratory route. It occurs during coughing, talking,
breathing or sneezing by an infected person on another person. Nose, mouth and
eyes are the loci through which microorganisms enter the body as they suspend
in warm and moist droplets. Diseases that spread through droplet infection are as
follows: meningitis, chickenpox, common cold, influenza, mumps, sore throat,
tuberculosis, measles, Rubella and whooping cough. Viral diseases also spread
by coughing or sneezing. Examples of these are common cold, influenza A and
B, mumps, measles and Rubella.
• (ii) Faecal–Oral Transmission:
• This type of transmission occurs when a person drinks faecal contaminated
water or eats contaminated food. Through this route, direct contact rarely occurs.
Usually infection occurs through the indirect routes. Sewage is released into a
drinking water supply and contaminates the water. This acts as mode of
transmission for the infectious agents of diseases like cholera, hepatitis A, polio,
typhoid and other similar diseases.
• (iii) Sexual Transmission:
• Transmission of disease can also occur during sexual activity with another
person, which includes vaginal, anal or oral sex. Infection can also occur from
secretions such as semen or the fluid secreted by the excited female which carry
infectious agents that get into the partner’s bloodstream through tiny tears in the
penis, vagina or rectum. Anal sex is thought to be more hazardous since the
penis causes more tears in the rectum than in the vagina. Some diseases
transmitted by the sexual route are HIV/AIDS, Chlamydia, genital warts,
gonorrhoea, hepatitis B, syphilis, herpes and trichomoniasis.
• (iv) Oral Transmission:
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• Diseases can also be transmitted primarily by oral means. These diseases can
be spread through direct oral contact such as kissing, or by indirect contact such
as by sharing a drinking glass or a cigarette. Diseases that are known to be
transmissible by kissing or by other direct or indirect oral contact comprise
Cytomegalovirus infections, Herpes simplex virus and infectious mononucleosis.
• (v) Transmission by Direct Contact:
• Contagious diseases are the diseases that can be transmitted by direct
contact. These diseases are common in schools and can be transmitted by
sharing objects of daily use such as a towel or clothes (called fomites), if they are
not washed thoroughly between uses. Some of the diseases that are
transmissible
by direct contact are Athlete’s foot, impetigo and syphilis.
• (vi) Vertical Transmission:
• This type of transmission occurs from the mother to the child, often in utero or
during childbirth. It is also called perinatal infection. It can also occur through
breast milk. Diseases which can be transmitted in this way include HIV, hepatitis
B and syphilis.
• (vii) Vector-Borne Transmission:
• Transmission can also occur through a vector. A vector is an organism that
does not cause disease itself but transmits infection by transferring pathogens
from one host to another.
Q 3
10
Describe the morphology and mode of multiplication of bacteriophages.
( Unit 10 ; Section 10.2 )
A 3
Morphology and mode of multiplication of bacteriophages
• Bacteriophages are viruses that infect and parasitize bacteria
10
• Phages occur widely in the environment such as sewage, faeces, soil and
other natural sources of mixed bacterial growth.
• T-even phages are tadpole-shaped and possess a head and a tail
• All the phages contain a nucleic acid enclosed in a capsid, which is made up of
capsomers.
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• Bradley (1967) described the following six morphological groups of
bacteriophages (Type A-F)
Multiplication
• Phages exhibit two different types of life cycle: lytic and lysogenic cycle
(i) Lytic Cycle
Replication of a virulent phage can be divided into five stages––adsorption,
penetration, synthesis of phage components, maturation and release of progeny
phages.
(ii) Lysogenic Cycle
In lysogenic cycle, the bacteriophage nucleic acid becomes inserted into the
bacterial chromosome.
Q 4
10
Explain the various techniques to diagnose viral infections under laboratory
conditions.
( Unit 8 ; Section 8.4 )
A 4
Techniques to diagnose viral infections
(i) Microscopy:
• The microscopic examination of stained smears is now employed for the
demonstration of virus elementary bodies.
(ii) Isolation and Characterization of Causative Virus:
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• This process requires a minimum period of one week and is expensive.
Direct Demonstration of Viral Antigen or Viral Nucleic Acid in Tissue,
(iii) Secretions or Excretions:
• This includes demonstration of viron, viral antigen and viral nucleic acid.
(iv) Detection and Measurement of Specific Antibodies:
• Availability of microtitre plates, monoclonal antibodies automation,
development of kits like latex agglutination and assays for IgM antibodies have
resulted in a revolution in approach to diagnostic serology for viral infections of
human beings.
(v) Molecular Diagnosis:
• Molecular methods such as probes and polymerase chain reaction provide
rapid, sensitive and specific
information about the presence of viruses in clinical samples.
Q 5
10
Describe the morphology, cultural characteristics, biochemical reactions,
pathogenesis, and laboratory diagnosis of Pneumococcus.
( Unit 2 ; Section 2.4 )
A 5
Morphology
Pneumococcus is lanceolate-shaped (flame shaped) and encapsulated. Small,
slightly elongated cocci, arranged in pairs (diplococci) with broad ends in
apposition are visible in Gram stain of smears from clinical specimens.
2
Cultural characteristics
Culture is carried out on blood agar and plates are aerobically incubated at 37°C
for 24 hours with 5–10% CO2. This results in small, shiny, dome-shaped and
translucent colonies, which are surrounded by alpha
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haemolysis .
On prolonged incubation, these colonies become depressed in the centre with an
elevated rim. This happens due to autolysis of the bacteria in older colonies.
Biochemical reactions
The test strain is inoculated into the insulin sugar medium with serum and is
incubated overnight at 37°C. Positive test is indicated by change of colour of the
media from red to yellow.
• These are sensitive to optochin and are soluble in bile salts. Individual cells are
between 0.5 and 1.25 μm in diameter.
• They do not form spores and are non-motile.
• They are catalase-negative and ferment glucose to lactic acid.
• Unlike other streptococci, they do not exhibit the M protein.
• The swelling reaction (also called as “quellung reaction”) is a sero-typing
technique that relies on the swelling of the capsule upon binding of homologous
antibody.
2
Pathogenesis
• Pneumococci produce disease by multiplying in the tissues.
2
• Produce no toxins but virulence of the organism is a function of its capsule.
This prevents ingestion by phagocytes.
2
Laboratory diagnosis
(A) Stained Smears
(B) Capsule Swelling Tests
(C) Culture
(D) Bile Solubility Test
(E) Optochin Sensitivity Test
Q 6
10
Discuss Measles virus under structure, pathogenesis, complication and
laboratory diagnosis.
( Unit 14 ; Section 14.4 )
A 6
Structure
• The virus resembles paramyxoviruses in morphology. It is spherical and about
120-250 nm in diameter. It has tightly coiled helical nucleocapsid surrounded by
lipoprotein envelope having haemagglutinin spikes, but neuraminidase spikes are
absent.
2
Pathogenesis
Measles virus is acquired by inhalation. Incubation period varies from 10 to 12
days. The virus multiplies in lymphoid tissue of respiratory tract and invades the
bloodstream (primary viraemia). The virus spreads to the reticulo-endothelial
system through blood. After multiplying there, a secondary viraemia occurs. The
virus is then transported to the epithelial surfaces including the skin, mouth,
respiratory tract and conjunctiva. It is characterized by high fever, cough and
conjunctivitis. Koplik's spots can be seen on the buccal mucosa and are
pathognomonic of measles.
2
Complication
(i) Measles decreases the resistance of the respiratory epithelium; therefore,
patients may develop secondary bacterial infections such as otitis media middle
ear infection), bronchopneumonia (Bronchopneumonia is an acute or
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chronic inflammation of the lungs)and croup (Croup or laryngotracheobronchitis)
is a respiratory condition that is usually triggered by an acute viral infection of the
upper airway).
(ii) Giant cell pneumonia may occur in a person who has impaired cellmediated
immunity.
(iii) Post-measles encephalitis and subacute sclerosing panencephalitis (SSPE)
may also occur (Subacute sclerosing panencephalitis (SSPE) is a progressive,
debilitating, and deadly brain disorder related to measles
(rubeola) infection).
Laboratory diagnosis
(i) Direct demonstration: Multinucleated giant cell can be demonstrated in
Giemsa-stained smears of nasal secretions.
(ii) Isolation: The measles virus can be isolated, with some difficulty, from throat
washing, blood, nasopharyngeal swab and conjunctiva during the prodromal
phase and up to about 2 days after appearance of the rash. Virus may be
obtained from the urine for a few more days.
(iii) Serology: Measles-specific IgM antibody in the patient's serum can be
detected by ELISA.
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