Notes of lessonDepartment

of BIOMEDICAL

T.A.SELVA KUMAR

IV Semester

Pathology & Microbiology

BM2252

HYPERSENSITIVITY

Hypersensitivity refers to undesirable (damaging, discomfort-producing and sometimes fatal) reactions produced by the normal immune system.

Hypersensitivity reactions require a pre-sensitized (immune) state of the host.

The four-group classification was expounded by P. H. G. Gell and Robin Coombs in 1963.

Comparison table

Type Alternative names

Often mentioned disorders

Mediators

1 Allergy Atopy

Anaphylaxis

Asthma

IgE

2 Cytotoxic, antibody-dependent

erythroblastosis fetalis

Goodpasture's syndrome

autoimmune hemolytic anemia

IgM or IgG

(Complement)

3 Immune complex disease

Serum sickness

Arthus reaction

SLE

IgG (Complement)

4 cell-mediated contact dermatitis

tuberculosis

Chronic transplant rejection

Cell-mediated

Type 1 – Immediate (or atopic, or anaphylactic)

• Type 1 hypersensitivity is an allergic reaction provoked by reexposure to a specific type of antigen referred to as an allergen.

• Exposure may be by ingestion, inhalation injection, or direct contact. The difference between a normal immune response and a type I hypersensitive response is that plasma cells secrete IgE.

• This class of antibodies binds to Fc receptors on the surface of tissue mast cells and blood basophils.

• Mast cells and basophils coated by IgE are "sensitized." • Later exposure to the same allergen, cross-links the bound IgE

on sensitized cells resulting in degranulation and the secretion of pharmacologically active mediators such as histamine, leukotriene, and prostaglandin that act on the surrounding tissues.

• The principal effects of these products are vasodilation and smooth-muscle contraction.

The reaction may be either local or systemic. Symptoms vary from mild irritation to sudden death from anaphylactic shock.Treatment usually involves epinephrine, antihistamines, and corticosteroids.If the entire body gets involved, then anaphylaxis can take place; an acute, systemic reaction that can prove fatal.

Some examples:

Allergic asthma Allergic conjunctivitisAllergic rhinitis ("hay fever") Anaphylaxis Angioedema Urticaria (hives) Eosinophilia Penicillin

Cephalosporin

Type 2 - Antibody-dependent

• In type 2 hypersensitivity, the antibodies produced by the immune response bind to antigens on the patient's own cell surfaces.

• The antigens recognized in this way may either be intrinsic ("self" antigen, innately part of the patient's cells) or extrinsic (absorbed onto the cells during exposure to some foreign antigen, possibly as part of infection with a pathogen).

• These cells are recognised by macrophages or dendritic cells which act as antigen presenting cells, this causes a B cell response where antibodies are produced against the foreign antigen.

• An example here is the reaction to penicillin where the drug can bind to red blood cells causing them to be recognised as different, B cell proliferation will take place and antibodies to the drug are produced.

• IgG and IgM antibodies bind to these antigens to form complexes that activate the classical pathway of complement activation for eliminating cells presenting foreign antigens (which are usually, but not in this case, pathogens).

• That is, mediators of acute inflammation are generated at the site and membrane attack complexes cause cell lysis and death. The reaction takes hours to a day.

• Another form of type 2 hypersensitivity is called antibody-dependent cell-mediated cytotoxicity (ADCC).

• Here, cells exhibiting the foreign antigen are tagged with antibodies (IgG or IgM).

• These tagged cells are then recognised by natural killer (NK) cells and macrophages (recognised via IgG bound (via the Fc region) to the effector cell surface receptor, CD16 (FcγRIII)), which in turn kill these tagged cells.

Some examples:• Autoimmune hemolytic anemia • Goodpasture's syndrome • Pemphigus • Pernicious anemia (if autoimmune) • Immune thrombocytopenia • Transfusion reactions • Hashimoto's thyroiditis • Graves disease (see type V below) • Myasthenia gravis (see type V below) • Rheumatic fever • Hemolytic disease of the newborn (erythroblastosis fetalis) • Acute transplant rejection

Type 3 - Immune complex

• Type 3 hypersensitivity occurs when antigens and antibodies are present in roughly equal amounts, causing extensive cross-linking.

• Large immune complexes that cannot be cleared are deposited in vessel walls and induce an inflammatory response.

• The reaction can take hours, days, or even weeks to develop.

Some clinical examples:

• Rheumatoid arthritis • Immune complex glomerulonephritis • Serum sickness • Subacute bacterial endocarditis • Symptoms of malaria • Systemic lupus erythematosus • Arthus reaction • Farmer's lung (Arthus-type reaction) • Polyarteritis nodosa

Type 4 - Cell-mediated (delayed-type hypersensitivity, DTH)

• Type 4 hypersensitivity is often called delayed type as the reaction takes two to three days to develop.

• Unlike the other types, it is not antibody mediated but rather is a type of cell-mediated response.

• CD8+ cytotoxic T cells and CD4+ helper T cells recognise antigen in a complex with either type 1 or 2 major histocompatibility complex.

• The antigen-presenting cells in this case are macrophages which secrete IL-12, which stimulates the proliferation of further CD4+ T cells.

• CD4+ T cells secrete IL-2 and interferon gamma, further inducing the release of other Type 1 cytokines, thus mediating the immune response.

Activated CD8+ T cells destroy target cells on contact while activated macrophages produce hydrolytic enzymes and, on presentation with certain intracellular pathogens, transform into multinucleated giant cells

Some clinical examples:Contact dermatitis (poison ivy rash, for example)

Atopic dermatitis (eczema) Temporal arteritis Symptoms of leprosy Symptoms of tuberculosis Mantoux test Coeliac disease

Organization and Structure of Microorganisms

• Phylogenetic relationships amongst different cell types

• Based on ribosomal RNA sequence comparsions (16S, 23S)

• 3 basic groups or domains established (domains are a higher order than kingdoms, ie are superkingdoms)

• The 3 domain = Bacteria, Archaea and Eucarya • 3 domains are related to each other; progenote =

hypothetical ancient universal ancestor of all cells. • Natural relationships amongst cells established

(phylogeny)

Diagramatic representation of cells

• Microbes have different shapes and is of advantage• Cell wall establishes the shape of a microbial cell but environmenta

conditions can change it • Shapes include:

– Spheres called cocci (greek = berry) can divide once in one axis to produce diplococci (Neisseria gonnorrhoeae, N. meningitidis), or more than once to produce a chain (Streptococcus pyogenes), divides regularly in two planes at right angles to produce a regular cuboidal packet of cells (xxx) or in two planes at different angles to produce a cluster of cells (Staphyloccus aureus)

– Cylinders called rods or bacilli (Latin bacillus = walking stick) – Spiral or spirilli (Greek spirillum = little coil)

• Shape offers an advantage to the cell: – Cocci: more ressistant to drying than rods – Rods: More surface area  & easily takes in dilute nutrients from the

environment – Spiral: Corkscrew motion & therefore less ressistant to movement – Square: Assists in dealing with extreme salinities

Virus structure • At the simplest level, the function of the outer shells (CAPSID) of a

virus particle is to protect the fragile nucleic acid genome from: • Physical damage - Shearing by mechanical forces. • Chemical damage- UV irradiation (from sunlight) leading to

chemical modification. • Enzymatic damage - Nucleases derived from dead or leaky cells or

deliberately secreted by vertebrates as defence against infection. • The protein subunits in a virus capsid are multiply redundant, i.e.

present in many copies per particle. Damage to one or more subunits may render that particular subunit non-functional, but does not destroy the infectivity of the whole particle. Furthermore, the outer surface of the virus is responsible for recognition of the host cell. Initially, this takes the form of binding of a specific virus-attachment protein to a cellular receptor molecule. However, the capsid also has a role to play in initiating infection by delivering the genome from its protective shell in a form in which it can interact with the host cell.

MULTIPLICATION OF VIRUS

GROWTH OF BACTERIA

Culture Media for the Growth of Bacteria • The biochemical (nutritional) environment is made available as a

culture medium, and depending upon the special needs of particular bacteria (as well as particular investigators) a large variety and types of culture media have been developed with different purposes and uses.

• A chemically-defined (synthetic) medium (Table 4a and 4b) is one in which the exact chemical composition is known.

• A complex (undefined) medium (Table 5a and 5b) is one in which

the exact chemical constitution of the medium is not known

• A defined medium is a minimal medium (Table 4a) if it provides only the exact nutrients (including any growth factors) needed by the organism for growth.

A selective medium is one which has a component's) added to it which will inhibit or prevent the growth of certain types or species of bacteria and/or promote the growth of desired species.

A culture medium may also be a differential medium if allows the investigator to distinguish between different types of bacteria based on some observable trait in their pattern of growth on the medium.

An enrichment medium (Table 5a and 5b) contains some component that permits the growth of specific types or species of bacteria, usually

because they alone can utilize the component from their environment.

Physical and Environmental Requirements for

Microbial Growth

• The Effect of Oxygen

• The Effect of Temperature on Growth

• The Effect of pH on Growth

• Water Availability

Bacterial Growth Curve

• A growth curve in biology generally concerns a measured property such as population size, body height or biomass. Values for the measured property can be plotted on a graph as a function of time.

• Bacterial Growth Curve: The schematic growth curve shown below is

associated with simplistic conditions known as a batch culture. It refers to a single bacterial culture, introduced into and growing in a fixed volume with a fixed (limited) amount of nutrient. Industrial situations involving MIC tend to be much more complex in nature than such a simplified model.

Bacterial Growth Curve

• Lag Phase: Bacteria are becoming "acclimated" to the new

environmental conditions to which they have been introduced (pH, temperature, nutrients, etc.). There is no significant increase in numbers with time.

• Exponential Growth Phase: The living bacteria population increases rapidly with

time at an exponential growth in numbers, and the growth rate increasing with time. Conditions are optimal for growth.

• Stationary Phase: With the exhaustion of nutrients and build-up of

waste and secondary metabolic products, the growth rate has slowed to the point where the growth rate equals the death rate. Effectively, there is no net growth in the bacteria population.

• Death Phase: The living bacteria population decreases with time,

due to a lack of nutrients and toxic metabolic by-products.

MICROSCOPY

Microscopy

• Resolving Power

• measures the ability to distinguish small objects close together • r.p. = 0.61 (lambda)

____________

(N sinØ)

Where lambda = wavelength of illuminating light.

Microscopy

• Resolving Power

• measures the ability to distinguish small objects close together • r.p. = 0.61 (lambda)

____________

(N sinØ)

Where lambda = wavelength of illuminating light.

• R.P. is smallest for violet light, but because human eye is more sensitive to blue, optimal R.P. is achieved with blue light (~450 nm). Use filters to remove other light in best microscopes

• n sinØ is called numerical aperture. It measures how much light cone spreads out between condenser & specimen. More spread = better resolution. Ø = angle of light cone; maximum value is 1.0

• n = refractive index. n = 1.0 in air. Can increase with certain oils (up to 1.4), called immersion oil. N.A. is property of lens. Look on side of lens to identify.

• Theoretical limit of R.P. for light scope is 0.2 micrometers.

Optical Instrument

Resolving Power RP in Angstroms

Human eye 0.2 millimeters (mm)

2,000,000 A

Light microscope 0.20 micrometers (µm)

2000A

Scanning electron microscope (SEM)

5-10 nanometers (nm)

50-100A

Transmission electron microscope

(TEM)

0.5 nanometers (nm)

5A

Bright Field Microscopy

• With a conventional bright field microscope, light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. We see objects in the light path because natural pigmentation or stains absorb light differentially, or because they are thick enough to absorb a significant amount of light despite being colorless. A Paramecium should show up fairly well in a bright field microscope, although it will not be easy to see cilia or most organelles. Living bacteria won't show up at all unless the viewer hits the focal plane and distorts the image by using maximum contrast.

Bright Field Microscopy

Electron Microscope

• Physicists discovered electrons have wave properties. Can use magnetic coils like lenses to focus beams of electrons. Basic design of EM similar to light scope

• But: electrons don't scatter from H, C, O, N: must add heavy atoms (e.g. Pb, Ur, Os, Gold) as stains.

• Also, electrons are scattered by air molecules. So must remove air from microscope with vaccum pump. But water in specimen will evaporate, so must be removed by dehydration after fixation. Cannot view living specimens.

Transmission Electron Microscope (TEM)

• See slide. R.P. approx. 1000x better than light; 0.2 nm, instead of 0.2 micrometers.

• Excellent for seeing internal detail. But cannot use with large/thick specimens.

• Specimen Preparation: specimen must be thin. Use grids with thin film supports. Prepare thick materials by sectioning with glass knives sections about 20-100 nm thick. Prepare small preparations (viruses, or subcellular particles) by negative staining.

Transmission Electron Microscope (TEM)

Scanning Electron Microscope (SEM)

• Same principle as TV screen, except reflected (secondary) electrons used to produce magnified image.

• complementary to TEM. Only see surface view --no internal detail visible.

Infinite depth of focus, in contrast to light scopes.

• R.P. around 2 nm at best, usually a bit poorer. (100x better than light scope, not as powerful as TEM)

• Specimen Preparation: fix & dry specimen. Shadow with thin metal film (e.g. gold). Mount on block and scan. (Note: sometimes possible to use ordinary air-dried material; but charge builds up on surface, distorts image).

Scanning Electron Microscope (SEM)

Staining technique

Staining technique

• Staining is a biochemical technique of adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. It is similar to fluorescent tagging.

• Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes. Stains may be used to define and examine bulk tissues (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells, for instance), or organelles within individual cells.

• Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis.

Gram staining

PRINCIPLE:

• Both Gram-positive (Gm+) and Gram-negative (Gm) organisms form a complex of crystal violet and iodine within the bacterial cell during the Gram-staining procedure. Gm+ organisms are thought to resist decolorization by alcohol or acetone because cell wall permeability is markedly decreased when it is dehydrated by these solvents. Thus, the dye complex is entrapped within the cell, resist being washed out by the solvents, and Gm+ bacteria remain purple following this differential stain.

• In contrast, cell wall permeability of Gm- organisms is increased by ethyl alcohol washing because it removes the outer membrane from the Gram-negative cell wall. This allows the removal of the crystal violet-iodine complex from within the cell. The decolorized Gm- cell can then be rendered visible with a suitable counterstain, in this case Safranin O, which stains them pink. Pink which adheres to the Gm+ bacteria is masked by the purple of the crystal violet.  

REAGENTS FOR THE GRAM STAIN: Crystal violet (Hucker's Stain)

Gram's iodine: Dissolve 0.33 g of iodine and 0.66 g of potassium iodide in 100 mL of distilled water.

            Alternately, dilute 0.1 N iodine 1:4. (Gram's Iodine solution should be fresh. If it has weakened and appears tan it will not work.)

Ethyl alcohol (95%)

Counterstain stock solution : Dissolve 2.5 g of certified safranin 0 in 100 mL of 95% ethyl alcohol.

Counterstain working solution:  Dilute stock solution 1:10 with dH2O.

Procedure

• After the smear has been dried, heat-fixed, and cooled off, proceed as follows:

• Place slide on staining rack and cover specimen with crystal violet. Let stand for 1 minute.

• Wash briefly in tap water and shake off excess.

• Cover specimen with iodine solution and let stand for 1 minute.

• Wash with water and shake off excess.

• Tilt slide at 45° angle and decolorize with the acetone-alcohol solution until the purple color stops running. Wash immediately with water and shake off excess.

• Cover specimen with safranine and let stand for 30 seconds to 1 minute.

• Wash with water, shake off excess, and gently blot dry. The smear is now ready to be read. (Use oil immersion lens.)

Gram staining

Acid fast staining

• Purpose:  To differentiate between acid-fast and non acid-fast bacteria.• Principle:  Some bacteria contain a waxy lipid, mycolic acid, in there cell

wall.  This lipid makes the cells more durable and is commonly associated with pathogens.  Acid fast cell walls are so durable that the stain (carbol fuschin) must be driven into the cells with heat.  The cells are then decolorized with acid-alcohol, all other cells will decolorize with this strong solvent, but acid fast bacteria will not.  Other cells are then counterstained with methylene blue.

• Procedure:• 1. Deparaffinize and hydrate to distilled water.• 2. Carbol-fuchsin solution, microwave 80 power, 45 seconds, allow slides

to stand in hot solution for 5 minutes. Filter solution once a week.• 3. Wash in running tap water.• 4. 1% Acid alcohol until light pink and color stops running.• 5. Wash in running tap water for 5 minutes.• 6. Rinse in distilled water.• 7. Working methylene blue for 30 seconds.• 8. Rinse in water. Dehydrate, clear, and coverslip.

Acid fast staining

Capsular stainingProcedure:

• 1.With a wax pencil, label the left-hand comer of a clean glass slide with the name of the bacterium that will be stained.

• 2. As shown in figure 15.3, aseptically transfer a loopful of culture with an inoculating loop to the slide. Allow the slide to air dry. Do not heat-fix!

• 3. Place the slide on a staining rack. Flood the slide with crystal violet and let stand for 4 to 7 minutes.

• 4. Rinse the slide thoroughly with 20% copper sulfate.

• 5. Blot dry with bibulous paper.

• 6. Examine under oil immersion (a coverslip is not necessary) and draw the respective bacteria in the report for exercise 12. Capsules appear as faint blue halos around dark blue to purple cells.

Capsular staining

Flagellar staining• Procedure:

• 1. With a wax pencil, mark the left-hand comer of a clean glass slide with the name of the bacterium.

• 2. Aseptically transfer the bacterium with an inoculating loop from the turbid liquid at the bottom of the slant to 3 small drops of distilled water in the center of a clean slide that has been carefully wiped off with clean lens paper. Gently spread the diluted bacterial suspension over a 3 cm area using the inoculating needle .

• 3. Let the slide air dry for 15 minutes.

• 4. Cover the dry smear with solution A (the mordant) for 4 minutes.

• 5. Rinse thoroughly with distilled water.

• 6. Place a piece of paper toweling on the smear and soak it with solution B (the stain). Heat the slide in a boiling water bath for 5 minutes in an exhaust hood with the fan on. Add more stain to keep the slide from drying out.

• 7. Remove the toweling and rinse off excess solution B with distilled water. Flood the slide with distilled water and allow it to sit for 1 minute while more silver nitrate residue floats to the surface.

• 8. Then, rinse gently with water once more and carefully shake excess water off the slide.

• 9. Allow the slide to air dry at room temperature.

• 10. Examine the slide with the oil immersion objective. The best specimens will probably be seen at the edge of the smear where bacteria

are less dense.