Complement Fixation Test (CFT)

This test is based upon two properties of the complement viz:

a. Complent combines with all antigen-antibody complexes whether or not it is required for that reaction
b. Complement is needed in immunolytic reaction.

Test system

It contains an antigen and a serum suspected to be having antibody to that antigen. The serum is heat treated prior to the test to destroy its complement. Complement Is added in measured quantity to this system. This complement is the form of guinea pig serum which is considered a rich source of complement. The test system is incubated.

Indicator system

To test system, after incubation, is added the indicator system which consists of sheep
RBCs and antibody to sheep RBCs (haemolysin) and another incubation is allowed.
If there is specific antibody in the test system, it will bind to antigen and to this complex the complement will also get fixed. Hence, no complement will be available to combine with indicator system which though contains RBCs and their specific antibody, cannot undergo haemolysis unless complement gets attached. Absence of haemolysis shall indicated positive test or presence of specific antibody in the serum which has been added in the test system. Erythrocytes lysis is obtained in negative test.

Bacteria

A bacterial cell has a nuclear apparatus which is a loose arrangement of DNA This is surrounded cytoplasm which contains ribosomes, mesosomes and inclusion granules. The cytoplasm is enclosed within a cytoplasmic membrane. Bacterium has a rigid cell wall  Fimbriae and flagella are the surface adherents. Some bacteria may have a capsule (or loose slime) around the cell wall.

Shape and Size of Bacteria

The bacteria can be spheroidal (coccus), rod or cylindrical (bacillus) and spirillar (spirochaete). Very short bacilli are called as coccobacilli  Some of the bacilli may be curved or comma shaped (Vibrio cholerae).

Arrangement of Bacterial Cells

Streptococci are present in chains; staphylococci in grape-like clusters Cocci in pairs (diplococci) are suggestive of pneumococci, gonococci or menigococci.
Bacilli do not exhibit typical arrangement pattern except the Chinese letter arrangement shown by Corynebacterium diphtheriae

Surface Adherents and Appendages

CAPSULE The gels formed by the capsule adhere to the cell Capsule can be detected by negative staining ,with specific antiserum and observing the capsular swelling phenomenon called as Quellung reaction
Usually weakly antigenic Capsule production is better in vivo as compared to in vitro environment.
Eg. Capsules seen in Pneumococci,  Klebsiella, Escherichia coli, Haemophilus influenzae

Flagella : provide motility to the bacterium. 
Motile organisms: vibrios, pseudomonas, Esch.coli, salmonellae, spirochaetes and spirilla. 
Pathogenic cocci are nomotile.
Flagella measure in length from 3 to 20 µm and in diameter from 0.01 to 0.0 13 µm.
 
Arrangement

Bacteria with one polar flagellum are known as monotrichous; 
Tuft of several polar flagellae is known as lophotrichous
Presence of  Flagellae at both the ends of organism is amphitrichous 
Flagellae distributed all over the surface of the bacterium, it is called peritrichous.
•    Filament is composed of a protein-flagellin. The flagellar antigen is called as H (Hauch) antigen in contrast to somatic antigen which is called as O (Ohne haunch)

PILI (fimbriae) : hair like structures help in attachment also called sex pilli, transfers genetic material through conjugation , Present in Certain Gram negative bacteria. Only Composed of protein pilin  
Gram positive bacterium that has pili is Cornebacterium renale

The Cell Wall

The cell wall of  bacteria is multilayered structure. The external surface of cell wall is smooth in Gram positive bacteria  Gram negative bacteria have convoluted cell surfaces. The average thickness of cell wall is 0.15 to 0.50 .µm. Chemically composed of mucopeptide scaffolding formed by N acetyl glucosamine and N acetyl muramic acid
The cell wall is a three layered structure in Gram negative bacteria: outer membrane middle layer and plasma membrane. The outer membrane consists of lipoprotein and 1ipoppolysaccaride component

Functions of bacterial cell wall

 Provides shape , Gives rigidity , Protection, Surface has receptor sites for phages, Site of  antibody action,  Provides attachment to complement, Contains components toxic to host
 
Cytoplasmic Structures

The Plasma Membrane: This delicate membrane separates rigid cell wall from cytoplasm. It accounts for 30% of total cell weight. Chemically, it is 60% protein, 20-30% lipids and remaining carbohydrates.

 Mesosomes: 
 
 Principal sites of respiratory enzyme , Seen well in Gram positive bacteria as compared to Gram negative batcteria. Attachement of mesosomes to both DNA chromatin and membrane have been noticed thus help in cell division
 
Ribosomes: 

sites of protein synthesis. These are composed of RNA and proteins and constitute upto 4 of total cell protein and 90% of total cellular RNA.
Cytoplasmic Granules: Glycogen  :  Enteric bacteria
Poly-beta & hydroxy Butyrate : Bacillus & Pseudomonas
Babes-Ernst  :Corynebacterium & Yersinia pestis

Nuclear Apparatus

Bacterial DNA represents 2-3% of the cell weight and 10% of the volume of bacterium. Nucleous can be demonstrated by staining it with DNA specific Fuelgen stain .Consists of a single molecule of  double stranded DNA arranged in a circular form. Bacterial chromosome is haploid and replicates by binary fission, the bacteria may have  plasmid an extrachromosomal genetic material.
 

NUTRITION OF BACTERIA

Nutrients

Chemoheterotrophs: nutrient source is organic material
Bacteria also requires a source of  minerals.

Oxygen

On this basis bacteria have been divided into four groups.

Obligate Anaerobes: These grow only under conditions of high reducing intensity. These bacteria catalase peroxidase, superoxide dismutase and cytochrome systems
Clostridium and Bacteroides are important examples.

Facultalive Anaerobes. These can grow under both aerobic and anaerobic conditions and include members of family enterobacteriaceae and many other bacteria.

Obligatory Aerobes. These cannot grow unless oxygen is present in the medium. Pseudomonas belong to this group.

Microaerophillic. These organisms can grow under conditions with low oxygen tension. Clostridium tetani is an important example.
The strict anaerobes are unable to grow unless Eh is as low as 0.2 volt

Temperature

•    On the basis of temperature requirements, three groups of bacteria are recognised.

•    Psychrophilic : Growth in  the range of —5 to 30°C with an optimum of 10-20 

•    Mesophillic : bacteria grow best at 20-40°C with a range of 10-45°C. 

•    Medically important bacteria belong to this group

•    Myco. leprae is one such important example and it can grow only at reduced temperature such as footpad of mouse

•    Thermophillic organisms prefer high temperature (25-80°C) for growth and yield maximum growth at 50-60°C

pH :  Most pathogenic bacteria require a pH of  7.2-7.6 for their own optimal growth.
 

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.

Variant Forms of Bacteria

Prortoplast ; surface is completely devoid of cell wall component,

Spheroplast : Some residual cell wall component is present 

Autoplast: protoplasts which are produced by the action of organisms’ own autolytic enzymes.

L Form: replicate as pleomorphic filtrable elements with defective or no cell wall These are designated as L forms after the Lister Institute where these were discovered by Klineberger-Nobel.

Bacterial Spores: Gram positive bacilli and actinomycetes form highly resistant and dehydrated forms which are called as endospores. The surrounding mother.cell which give rise to them is known as Sporangium. These endospores are capable of survival under adverse conditions
Structure :smooth walled and ovoid or spherical. 

In bacilli, spores usually fit into the normal cell diameter except in Clostridium where these may cause a terminal bulge. (drum stick ) or central. , these look like areas of high refractilitv under light microscope.

Germination : This is the process of converting a spore into the vegetative cell. It occurs in less than 2 hours and has three stages:Activation, Germination, Outgrowth
 

Cell Functions:
-> Autolysis

- degradative reactions in cells caused by indigenous intracellular enzymes – usually occurs after cell death
- Irreversible (along with Coagulative necrosis or infarcts) – reversible: fatty degeneration, & hydropic degeneration

-> Autolysin:
•    Ab causing cellular lysis in the presence of complement
•    Autolytic enzymes produced by the organism degrade the cell’s own cell wall structures

-> In the presence of cephalosporins & penicillins, growing bacterial cells lyse
•    W/o functional cell wall structures, the bacterial cell bursts

-> Heterolysis: cellular degradation by enzymes derived from sources extrinsic to the cell (e.g., bacteria)

-> Necrosis: sum of intracellular degradative reactions occurring after individual cell death w/in a living organism