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.
 

Neutralization Test

These are basically of two types:

•    Toxin neutralization
•    Virus neutralization

In toxin neutralization homologous anti-bodies prevent the biological effect of toxin as observed in vivo in experimental animals (e.g. detection of toxin of Clostridia and Corynebacterium diphthenae) or by in vitro method (e.g. Nagler’s method).

In virus neutralization test various methods are available by which identity of virus can be established as well as antibody against a virus can be estimated.

Measurement of Bacterial of Growth

A convenient method is to determine turbidity by photoelectric colorimeter or spectrophotometer. 
The cell number can be counted as total cell number as well as viable count. Viable Count Viable number of bacteria can be counted by inoculating the suspension onto solid growth medium and counting the number of colonies. Since each colony is the end product of one viable bacterium, their count gives the number of viable bacteria in the suspension.
Total number of bacteria can be ascertained in specially designed chambers such as Coulter counter.
 

CHEMICAL AGENTS

Chlorine and iodine are most useful disinfectant Iodine as a skin disinfectant and chlorine as a water disinfectant have given consistently magnificent results. Their activity is almost exclusively bactericidal and they are effective against sporulating organisms also. 
Mixtures of various surface acting agents with iodine are known as iodophores and these are used for the sterilization of dairy products.

Apart from chlorine, hypochlorite, inorganic chioramines are all good disinfectants but they act by liberating chlorine. 

Hydrogen peroxide in a 3% solution is a harmless but very weak disinfectant whose primary use is in the cleansing of the wound.
 
Potassium permanganate is another oxidising agent which is used in the treatment of urethntzs. 

Formaldehyde — is one of the least selective agent acting on proteins. It is a gas that is usually employed as its 37% solution, formalin. 

When used in sufficiently high concentration it destroys the bacteria and their spores.

Classification of chemical sterilizing agents

Chemical disinfectant

Interfere with membrane functions

•    Surface acting agents : Quaternary ammonium, Compounds, Soaps and fatty acids

•    Phenols : Phenol, cresol, Hexylresorcinol

•    Organic solvent : Chloroform, Alcohol

Denatures proteins

•    Acids and alkalies : Organic acids, Hydrochloric acid , Sulphuric acid

Destroy functional groups of proteins

•    Heavy metals :  Copper, silver , Mercury

•    Oxidizing agents: Iodine, chlorine, Hydrogen peroxide

•    Dyes : Acridine orange, Acriflavine

•    Alkylating agents : Formaldehyde, Ethylene oxide

Applications and in-use dilution of chemical disinfectants

Alcohols : Skin antiseptic Surface disinfectant, Dilution used 70%

Mercurials : Skin antiseptic Surface disinfectant Dilution Used 0.1 %

Silver nitrate : Antiseptic (eyes and burns)  Dilution Used 1 %

Phenolic compound : Antiseptic skin washes  Dilution Used .5 -5 %

Iodine : Disinfects inanimate object, Skin antiseptic Dilution used  2%

Chlorine compounds  : Water treatment Disinfect inanimate objects , Dillution used 5 %

Quaternary ammonium Compounds : Skin antiseptic , Disinfects inanimate object, Dilution Used < 1 %

Glutaraldehyde: Heat sensitve instruments, Dilution used 1-2 %

Cold sterilization can be achieved by dipping the precleaned instrument in 2% solution of gluteraldehyde for 15-20 minutes. This time is sufficient to kill the vegetative form as well as spores ofthe organisms that are commonly encountered in the dentistry.

Ethylene oxide is an a agent extensively used in gaseous sterilization. It is active against all kinds of bacteria and their spores. but its greatest utility is in sterilizing those objects which are damaged by heat (e.g. heart lung machine). It is also used to sterlise fragile, heat sensitive equipment, powders as well as components of space crafts.

Evaluation of Disinfectants

Two methods which are widely employed are:

 Phenol coefficient test, Kelsey -Sykes test
 
These tests determine the capacity of disinfectant as well as their ability to retain their activity.
 

ANTIGEN-ANTIBODY REACTIONS

Affinity of the antigen-antibody reaction refers to the intensity of the attraction between antigen and antibody molecule.
Antigen-antibody reactions

Reaction test            Modified test

Precipitation  -> Immunoelectrophoresis, Immunoprecipitation
Agglutination -> Latex agglutination, Indirect, Haemagglutination , Coagglutination ,Coombs test

Neutralization  -> Measurement of LD, Plaque assays

Complement fixation  -> Conglutination

Immunofluorescence ->  Indirect immunofiuorescence, Immunoofluoremetric Assay

Enzyme immunoassay -> Enzyme linked, Immunosorbent assay

Radioimmunoassay -> Immunoradiometric assay

Avidity is the strength of the bond after the formation of antigen-antibody complex.

Sensitivity refers to the ability of the test to detect even very minute quantities of antigen or antibody. A test shall be called as highly sensitive if false negative results are absent or minimal.

Specificity refers to the ability of the test to detect reactions between homologous antigens and antibodies only, and with no other. In a highly specific test, false positive reactions will be minimal or absent.

Immunoglobulin (Ig)

Immunoglobulins are glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that they migrate with globular proteins when antibody-containing serum is placed in an electrical field

FUNCTION
1. Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host.

2. The significant biological effects are a consequence of secondary "effector functions" of antibodies.Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn.

STRUCTURE OF IMMUNOGLOBULINS

The basic structure of the immunoglobulins is illustrated in figure 2. Although different immunoglobulins can differ structurally, they all are built from the same basic units.

A. Heavy and Light Chains

All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23kD) and two identical heavy chains (50-70kD)

B. Disulfide bonds

1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions The number of inter-chain disulfide bonds varies among different immunoglobulin molecules.

2. Intra-chain disulfide binds - Within each of the polypeptide chains there are also intra-chain disulfide bonds.

C. Variable (V) and Constant (C) Regions

When the amino acid sequences of many different heavy chains and light chains were compared, it became clear that both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. These are the:

1. Light Chain - VL (110 amino acids) and CL (110 amino acids)

2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)\(x = {-b \pm \sqrt{b^2-4ac} \over 2a}\)h the arms of the antibody molecule forms a Y. It is called the hinge region because there is some flexibility in the molecule at this point.

E. Domains

Three dimensional images of the immunoglobulin molecule show that it is not straight as depicted in figure 2A. Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond (figure 2B-D). These regions are called domains.

1. Light Chain Domains - VL and CL

2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)

F. Oligosaccharides

Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates may also be attached at other locations. 

IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS

Immunoglobulin fragments produced by proteolytic digestion –

A.  Fab 
Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 6. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.

Antigen binding – These fragments are  called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL. 

B. Fc 
Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.

Effector functions – The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment . 

Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites . This fragment is called F(ab’)2because it is divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab’)2binds antigen but it does not mediate the effector functions of antibodies.