Tuberculosis

1. Laboratory diagnosis of pulmonary tuberculosis

2. Classify Mycobacteria. Describe the pathogenesis and laboratory diagnosis of
pulmonary tuberculosis.
3. Describe the morphology of Mycobacterium tuberculosis. Write briefly on pathogenesis &
laboratory of pulmonary tuberculosis
4. Describe the pathogenicity and laboratory diagnosis of infections Caused by
Mycobacterium tuberculouosis
5. Describe the Morphology, Pathogenesis and Laboratory diagnosis of Pulmonary
tuberculosis
Leprosy
1. Lepromin test

Spirochetes
1. Classify spirochetes. Describe in detail the pathogenesis and laboratory diagnosis of
syphilis.

Clostridium
1. Lab diagnosis of gas gangrene
2. Classify clostridia and write about the morphology and cultural characters. Add a note on
prophylaxis of tetanus
3. Clostridium perfringens
4. Describe the morphology, cultural characteristics, etiopathogenesis and laboratory
diagnosis of Clostridium tetani
 Tuberculosis

Classification of Mycobacteria
Morphology of Mycobacterium tuberculosis
Pathogenesis of Pulmonary tuberculosis

● In the lung the bacteria meets with first line defense, the macrophages
● These macrophages engulf the organism, but they manages to survive
● Iternalization of bacilli triggers inflammatory reaction bringing other inflammatory cells
together forming a granuloma
● The granuloma in initial stages has a core of infected cells surrounded by inflammatory
cells
● Later cellular immunity develops, then the macrophages loaded with bacilli are
disintegrated forming caseous centre of the granuloma
● The bacteria still is dormant and may live for decades, this stage is called latent stage
and may persist throughout life in a person without causing infection
● If the immunity is good, the TB stops at this stage by resolution my be just leaving
behind few calcifications, on the other hand if the immunity is poor, the bacteria replicate,
escape from granuloma and go to other part of lung causing active tuberculosis
● From lungs it may spread to other organs causing miliary tuberculosis
Laboratory diagnosis of Pulmonary tuberculosis
What is Ziehl-Neelsen staining used for?
Ziehl Neelsen staining is routinely used as bacteriological staining method used to identify
acid-fast organisms, including Mycobacteria

The use of acid-alcohol in the technique earned it the name Acid-Fast Stain and the application
of heat in the technique gives it the name the hot method of Acid-Fast staining which is a
synonymous name for the Ziehl-Neelsen Staining technique.
This technique is used on microorganisms that are not easily stained by basic stains such as
Negative staining or Gram staining.
One of the most complex micro-organisms that require harsh treatment of the Ziehl-Neelsen
compounds is the Mycobacterium spp.
Mycobacterium, Actinomycetes, Cryptosporidium, some fungi etc contain a thick cell wall made
up of lipoidal complexes known as mycolic acid.
Mycolic acid is difficult to stain and therefore simple stains like gram staining can not penetrate
the thick cell wall of these organisms.
They require harsher treatments to allow stain penetration for identification and examination and
hence the use of the Ziehl-Neelsen or the hot method of Acid-fast stain.

Objectives of the Ziehl-Neelsen Staining

To differentiate between acid-fast bacilli and non-acid-fast bacilli.
To stain Mycobacterium species.

Principle of the Ziehl-Neelsen Staining

The Ziehl-Neelsen stain employs basic fuchsin and phenol to highlight Mycobacterium species'
cell walls. Mycobacteria's high lipid content, primarily mycolic acid, renders them impermeable
to simple stains. Carbol-fuchsin, aided by heat, penetrates this lipid-rich barrier. Acid alcohol
treatment solidifies the bond between the stain and the bacteria's components, ensuring
retention during washing. Acid-fast bacteria retain the red color of carbol-fuchsin, while
non-acid-fast bacteria appear blue after counterstaining with methylene blue. This method is
pivotal in identifying Mycobacterium tuberculosis and Mycobacterium leprae.
Lepromin test
A lepromin skin test is used to determine the type of Hansen’s disease (leprosy) a person has
contracted. The lepromin skin test is also called the leprosy skin test.
It is a delayed type of hypersensitive reaction

The negative reaction or late reaction takes place 1-2 weeks after the injection
Elisa test
ELISA stands for “enzyme-linked immunosorbent assay”. ELISAs is widely used as a research
method for quantifying proteins or antigens in an unknown solution, or in medicine as a
diagnostic tool. Used for testing blood samples for the presence of infectious disease
antibodies, testing the binding between a newly isolated antibody and its target protein, or a
variety of other tests that rely on Ag-Ab binding (antibody-antigen interactions).

Direct ELISA
Initially in a direct ELISA test which is considered to be the simplest type of ELISA the antigen is
adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin)
is added to block all the other binding sites. While an enzyme is linked to an antibody in a
separate reaction, the enzyme-antibody complex is applied to adsorb to the antigen. After
excess enzyme-antibody complex is washed off, enzyme-antibody bound to antigen is left. By
adding in the enzyme's substrate, the enzyme is detected illustrating the signal of the antigen.
Direct ELISA, when compared to other forms of ELISA testing, is performed faster because only
one antibody is being used and fewer steps are required.
1. Components of the sample are taken in a well
2. They are then added with antibody solution, in samples which contain the required
antigen, these antibody will bind to form a complex
3. Then they are washed
4. To this a substrate is given, the antibody-antigen comlex is conjugated to an enzyme
which has a capacity to convert this substrate to a detectable product which gives colour
to the solution

Indirect ELISA
It is a two-step ELISA which involves two binding process of primary antibody and labeled
secondary antibody. The primary antibody is incubated with the antigen followed by the
incubation with the secondary antibody.(Here the antigen is known and is placed in the well and
the antibody to be known is added into it.)
1. Micro-well plates are incubated with antigens
2. Samples with antibodies are added and washed.
3. Enzyme linked secondary antibody are added and washed.
4. A substrate is added, and enzymes on the antibody elicit a chromogenic or fluorescent signal.
If the colour is produced then it means there is presence of antibody against the known antigen

The sandwich ELISA quantify antigens between two layers of antibodies (i.e. capture and
detection antibody). The advantage of Sandwich ELISA is that the sample does not have to be
purified before analysis, and the assay can be very sensitive (up to 2 to 5 times more sensitive
than direct or indirect ELISA).
The principle is as follows:
(1) Plate is coated with a capture antibody;
(2) sample is added, and any antigen present binds to capture antibody;
(3) detecting antibody is added, and binds to antigen;
(4) enzyme-linked secondary antibody is added, and binds to detecting antibody;
(5) substrate is added, and is converted by enzyme to detectable form.
 Spirochetes

Classify spirochetes

Pathogenesis of syphilis
Primary syphilis
Congenital syphilis
Congenital syphilis (CS) is caused by transmission of the spirochete Treponema pallidum from
the mother to the fetus, resulting in a multitude of clinical presentations ranging from
asymptomatic, premature birth, and a wide array of clinical signs and symptoms to stillbirth.
Other features, frontal bossae, high arched palate,mulberry molars, saddle nose, thick clavicle,
Skin: Jaundice, rash and desquamation. Abdomen: Hepatosplenomegaly (enlarged liver
and spleen) Eye: Chorioretinitis and pigmentary chorioretinopathy (salt and pepper
type), glaucoma, cataracts, interstitial keratitis, optic neuritis.
Laboratory diagnosis of syphilis.
Venereal disease research laboratory (VDRL) test
A positive test result means the patient may have syphilis. If the test is positive, the next step is
to confirm the results with an FTA-ABS test, which is a more specific syphilis test. The VDRL
test's ability to detect syphilis depends on the stage of the disease.

RPR (rapid plasma reagin) is a screening test for syphilis. It measures substances (proteins)
that are present in the blood of people who may have the disease.
Fluorescent treponemal antibody absorption test. The FTA-ABS test is used to detect antibodies
to the bacteria Treponema pallidum, which causes syphilis. Using antibodies specific for the
Treponema pallidum species, such tests would be assumed to be more specific than
non-treponemal testing such as VDRL but have been shown repeatedly to be sensitive

Treponema Pallidum Hemagglutination (TPHA) test is used to detect antibodies in the blood
against syphilis

Treponema Palidum Immobilization Test

The test is performed in diluting pipettes of the type used in. making white blood cell counts.
The serum to be tested and treponemal suspension con- taining active complement are taken
up successively into the pipette, which is then closed. with a rubber ring, shaken andplaced in
the incubator.
 Clostridium
Lab diagnosis of gas gangrene
Classify clostridia
Morphology and cultural characters of clostridia
Clostridium perfringens
Clostridium tetani
Clostridium botulinum
Prophylaxis of tetanus
Laboratory diagnosis of Clostridium tetani
Clostridium perfringens

Etiopathogenesis Clostridium tetani