BACTERIAL GROWTH

The conversion of a parental cell into two daughters constitutes the bacterial life cycle and the time taken to complete cell cycle is known as generation_time. This is around 15 minutes in vegetative bacteria except mycobacteria.

Bacterial Growth Curve

In the presence of fresh growth medium a bacterium shows following four phases;

The Lag phase -> The Log phase -> The Stationary phase  -> The Decline phase

The Lag Phase : short duration , bacteria adapt themselves to new environment 

The Log Phase (Exponential Phase) : Regular growth of bacteria occurs The morphology of bacteria is best developed in this phase and organisms manifest typical biochemical characters. 

- Most of the cidal Abx work best in this phase
•    i.e. Ampicillin
- Best phase for staining bacterial cultures

Chemostat and turbidostat are examples of technique by which this phase can be prolonged.

Stationary Phase : balanced growth and cell division cannot be sustained. The total cell Count remains static till lysis supervenes, but the viable cell count quickly declines.

Decline Phase: death phase. Dyeing bacteria exceed the dividing bacterias.
 

PHAGOCYTOSIS AND INTRACELLULAR KILLING

A. Phagocytic cells

1. Neutrophiles/Polymorphonuclear cells

PMNs are motile phagocytic cells that have lobed nuclei. They can be identified by their characteristic nucleus or by an antigen present on the cell surface called CD66. They contain two kinds of granules the contents of which are involved in the antimicrobial properties of these cells. 

The second type of granule found in more mature PMNs is the secondary or specific granule. These contain lysozyme, NADPH oxidase components, which are involved in the generation of toxic oxygen products, and characteristically lactoferrin, an iron chelating protein and B12-binding protein.

2. Monocytes/Macrophages

 Macrophages are phagocytic cells . They can be identified morphologically or by the presence of the CD14 cell surface marker. 

B. Response of phagocytes to infection 

Circulating PMNs and monocytes respond to danger (SOS) signals generated at the site of an infection. SOS signals include N-formyl-methionine containing peptides released by bacteria, clotting system peptides, complement products and cytokines released from tissue macrophages that have encountered bacteria in tissue.
Some of the SOS signals stimulate endothelial cells near the site of the infection to express cell adhesion molecules such as ICAM-1 and selectins which bind to components on the surface of phagocytic cells and cause the phagocytes to adhere to the endothelium. 
Vasodilators produced at the site of infection cause the junctions between endothelial cells to loosen and the phagocytes then cross the endothelial barrier by “squeezing” between the endothelial cells in a process called diapedesis.

 Once in the tissue spaces some of the SOS signals attract phagocytes to the infection site by chemotaxis (movement toward an increasing chemical gradient). The SOS signals also activate the phagocytes, which results in increased phagocytosis and intracellular killing of the invading organisms.

C. Initiation of Phagocytosis 

Phagocytic cells have a variety of receptors on their cell membranes through which infectious agents bind to the cells. These include:

1. Fc receptors – Bacteria with IgG antibody on their surface have the Fc region exposed and this part of the Ig molecule can bind to the receptor on phagocytes. Binding to the Fc receptor requires prior interaction of the antibody with an antigen. Binding of IgG-coated bacteria to Fc receptors results in enhanced phagocytosis and activation of the metabolic activity of phagocytes (respiratory burst).

2. Complement receptors – Phagocytic cells have a receptor for the 3rd component of complement, C3b. Binding of C3b-coated bacteria to this receptor also results in enhanced phagocytosis and stimulation of the respiratory burst. 

3. Scavenger receptors – Scavenger receptors bind a wide variety of polyanions on bacterial surfaces resulting in phagocytosis of bacteria.

4. Toll-like receptors – Phagocytes have a variety of Toll-like receptors (Pattern Recognition Receptors or PRRs) which recognize broad molecular patterns called PAMPs (pathogen associated molecular patterns) on infectious agents. Binding of infectious agents via Toll-like receptors results in phagocytosis and the release of inflammatory cytokines (IL-1, TNF-alpha and IL-6) by the phagocytes.

D. Phagocytosis 

The pseudopods eventually surround the bacterium and engulf it, and the bacterium is enclosed in a phagosome. During phagocytosis the granules or lysosomes of the phagocyte fuse with the phagosome and empty their contents. The result is a bacterium engulfed in a phagolysosome which contains the contents of the granules or lysosomes.

E. Respiratory burst and intracellular killing

During phagocytosis there is an increase in glucose and oxygen consumption which is referred to as the respiratory burst. The consequence of the respiratory burst is that a number of oxygen-containing compounds are produced which kill the bacteria being phagocytosed. This is referred to as oxygen-dependent intracellular killing. In addition, bacteria can be killed by pre-formed substances released from granules or lysosomes when they fuse with the phagosome. This is referred to as oxygen-independent intracellular killing.

1. Oxygen-dependent myeloperoxidase-independent intracellular killing

During phagocytosis glucose is metabolized via the pentose monophosphate shunt and NADPH is formed. Cytochrome B which was part of the specific granule combines with the plasma membrane NADPH oxidase and activates it. The activated NADPH oxidase uses oxygen to oxidize the NADPH. The result is the production of superoxide anion. Some of the superoxide anion is converted to H2O2 and singlet oxygen by superoxide dismutase. In addition, superoxide anion can react with H2O2 resulting in the formation of hydroxyl radical and more singlet oxygen. The result of all of these reactions is the production of the toxic oxygen compounds superoxide anion (O2-), H2O2, singlet oxygen (1O2) and hydroxyl radical (OH•).

2. Oxygen-dependent myeloperoxidase-dependent intracellular killing 

As the azurophilic granules fuse with the phagosome, myeloperoxidase is released into the phagolysosome. Myeloperoxidase utilizes H2O2 and halide ions (usually Cl-) to produce hypochlorite, a highly toxic substance. Some of the hypochlorite can spontaneously break down to yield singlet oxygen. The result of these reactions is the production of toxic hypochlorite (OCl-) and singlet oxygen (1O2).

3. Detoxification reactions 

PMNs and macrophages have means to protect themselves from the toxic oxygen intermediates. These reactions involve the dismutation of superoxide anion to hydrogen peroxide by superoxide dismutase and the conversion of hydrogen peroxide to water by catalase. 

4. Oxygen-independent intracellular killing 

In addition to the oxygen-dependent mechanisms of killing there are also oxygen–independent killing mechanisms in phagocytes: cationic proteins (cathepsin) released into the phagolysosome can damage bacterial membranes; lysozyme breaks down bacterial cell walls; lactoferrin chelates iron, which deprives bacteria of this required nutrient; hydrolytic enzymes break down bacterial proteins. Thus, even patients who have defects in the oxygen-dependent killing pathways are able to kill bacteria. However, since the oxygen-dependent mechanisms are much more efficient in killing, patients with defects in these pathways are more susceptible and get more serious infections.

ANTIGEN-ANTIBODY REACTIONS

I. NATURE OF ANTIGEN-ANTIBODY REACTIONS

A. Lock and Key Concept 

The combining site of an antibody is located in the Fab portion of the molecule and is constructed from the hypervariable regions of the heavy and light chains. Antigen-antibody reactions is one of a key (i.e. the antigen) which fits into a lock (i.e. the antibody).

B. Non-covalent Bonds 

The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These include hydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds. 

C. Reversibility
Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible.
II. AFFINITY AND AVIDITY

A. Affinity 
Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody .

B. Avidity
Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities.

III. SPECIFICITY AND CROSS REACTIVITY

A. Specificity 

Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. In general, there is a high degree of specificity in antigen-antibody reactions. 

B. Cross reactivity 

Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen. 

Immunofluorescence

This is precipitation or complement fixation tests. The technique can detect proteins at concentrations of around 1 µg protein per ml body fluid. Major disadvantage with this technique is frequent occurrence of nonspecific fluorescence in the tissues and other material.
The fluorescent dyes commonly used are fluorescein isothocyanate (FITC). These dyes exhibit fluorescence by absorbing UV light between 290 and 495 nm and emitting longer wavelength coloured light of 525 nm which gives shining appearance (fluorescence) to protein labelled with dye. Blue green (apple green) fluorescence is seen with FITC and orange red with rhodamine.

Enzyme Immunoassays

These are commonly called as enzyme linked immunosorbent assays or EL1SA. It is a simple and versatile technique which is as sensitive as radioimmunoassays. It is now the
technique for the detection of antigens, antibodies, hormones, toxins and viruses.

Identification of organisms by immunofluorescence

Type of agent         Examples

Bacterial            Neisseria gonorrhoeae, H. influenzae ,Strept pyogenes, Treponema pallidum
Viral                  Herpesvirus, Rabiesvirus, Epstein-Barr virus
Mycotic             Candida albicans

Enzymatic activity results in a colour change which can be assessed visibly or quantified in a simple spectrophotometer.

Test for Antigen - Antibody Reactions

Antigens are those substance that stimulates the production of antibodies which, when enter into the body it reacts specifically in a manner that are clearly visible. 

Some antigens may not induce antibody production, but instead creates immunological tolerance. 
An antigen introduced into the body produces only specific antibodies and will react with only those specific antigens. 
These antibodies appear in the serum and tissue fluids. All antibodies are considered as immunoglobulin. They are mainly of five classes; IgG, IgA, IgM, IgD and IgE. 

Antigen- antibody reactions are known as serological reactions and are used as serological diagnostic tests for the identification of infectious diseases.

The reaction occurs mainly in three stages; 

1. The initial interaction between the antigen and antibody, which produces no visible effects. It is a reversible and rapid reaction.
2. The secondary stage leads to the demonstration proceedings, such as precipitation, agglutination, etc.
3. The tertiary reaction follows the neutralization or destruction of injurious antigens. These results in clinical allergy and other immunological diseases.

There are certain characteristics for antigen-antibody reactions;

1. Reaction is specific.
2. The whole molecules participate in the reaction, and not just a part of it.
3. No denaturation of antigen or antibody occurs during the reaction.
4. The combination usually occurs at the surface.
5. The combination is firm, but reversible
6. Agglutinins formed after agglutination usually are formed by both antigen and antibody together.
7. They can combine in varying proportions.

Measurement of antigen and antibody are made in terms of mass or as units or titre.

Serological reactions include;

1. Precipitation reaction

a soluble antigen combining with the specific antibody in the presence of electrolytes at a suitable temperature and pH forming insoluble precipitins.  Commonly used tests are ring test, slide test, tube test, immunodiffusion, etc.

Radial Immunodiffusion 

In radial immunodiffusion antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed .
This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples.

Immunoelectrophoresis 

In immunoelectrophoresis, a complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs .

This tests is used for the qualitative analysis of complex mixtures of antigens

This test can also be used to evaluate purity of isolated serum proteins.

Countercurrent electrophoresis

In this test the antigen and antibody are placed in wells punched out of an agar gel and the antigen and antibody are electrophoresed into each other where they form a precipitation line. 

2. Agglutination reaction 

when a particulate antigen is mixed with its antibody in the presence of electrolytes at a suitable temperature and pH, the particles are clumped or agglutinated. When the antigen is an erythrocyte the term hemagglutination is used.

Applications of agglutination tests

i. Determination of blood types or antibodies to blood group antigens.
ii. To assess bacterial infections
e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.

Passive hemagglutination 

The agglutination test only works with particulate antigens. However, it is possible to coat erythrocytes with a soluble antigen (e.g. viral antigen, a polysaccharide or a hapten) and use the coated red blood cells in an agglutination test for antibody to the soluble antigen . This is called passive hemagglutination. 
The test is performed just like the agglutination test.

Applications include detection of antibodies to soluble antigens and detection of antibodies to viral antigens.

Coomb's Test (Antiglobulin Test)

DIRECT ANTIGLOBULIN TEST (DAT)

The DAT is used to detect IgG or C3 bound to the surface of the red cell.  In patients with hemolysis, the DAT is useful in determining whether there is an immune etiology.  
A positive DAT can occur without hemolysis
Immune causes of hemolysis including autoimmune hemolytic anemias, drug induced hemolysis, and delayed or acute hemolytic transfusion reactions are characterized by a positive DAT.

INDIRECT ANTIGLOBULIN TEST (IAT)

The IAT (antibody screen) is performed by incubating patient serum with reagent screening red cells for approximately 20 minutes and then observing for agglutination.  If the antibody screen is positive, additional testing is required to determine the specificity of the antibody. 

The IAT is used to detect red cell antibodies in patient serum.  Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both.

3. Complement fixation test (CFT)

the ability of antigen antibody complexes to fix complement is made use in this test. Complement is something which takes part in any immunological reaction and absorbed during the combining of antigen with its specific antibody. 

The best example of CFT is the Wassermann reaction done for the detection of Syphilis.

4. Neutralization test

different types of these are available. Virus neutralization, toxin neutralization, etc. are some of its kind.

5. Opsonization

this makes use of the determination of opsonic index, which is the ratio of the phagocytic activity of patient’s blood to the phagocytic activity of the normal patient’s for a given bacterium.

6. Immunfluorescence 

the method of labeling the antibodies with fluorescent dyes and using them for the detection of antigens in tissues.

7. Radioimmunoassay (RIA)

 is a competitive binding radioisotopes and enzymes are used as labels to conjugate with antigens or antibodies.

8. Enzyme Immuno Assay (EIA)

 the assays based on the measurement of enzyme labeled antigen or antibody. The most common example is ELISA used to detect HIV.

9. Immunoelectroblot

 it uses the sensitivity of Enzyme immunoassay with a greater specificity. Example is Western blot done for the serodiagnosis of HIV infection.

Classification:

Neutrophiles (pH = 7.0)
- P. aeruginosaqo
- Clostridium sporogenes
- Proteus species

Acidophiles (pH < 7.0)
- Thiobacillus thiooxidans
- Sulfollobus acidocaldaarius
- Bacillus acidocaldarius

Alkaliphiles (pH > 7.0)
- Nitrobacter species
- Streptococcus pneumoniae