DISINFECTION AND STERILIZATION

•    Sterilization is the best destruction or com removal_of all forms of micro organisms.
•    Disinfection is the destruction of many microorganisms but usually the b spores.
•    Antisepsis is the destruction or inhibition of microorganisms in living tissues thereby limiting or preventing the harmful effect of infection.
•    Astatic Agent  would only inhibit the growth of microorganisms (bacteriostatic, fungistatic, sporostatic).
•    Acidal agent would kill the microorganism (bactericidal. virucidal, fungicidal)
•    Sterilants are the chemicals which under controlled conditions can kill sporinQ bacteria.
 

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.

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.

COMPLEMENT

The complement system primarily serves to fight bacterial infections. 

The complement system can be activated by at least three separate pathways. 
1) alternative pathway -
- The alternative pathway of complement activation starts with the spontaneous hydroysis of an internal thioester bond in the plasma complement component C3 to result in C3(H2O).

- The smaller cleavage products C3a, C4a, C5a, sometimes called "anaphylatoxins", act as phagocytes, they cause mast cell degranulation and enhance vessel permeability, thereby facilitating access of plasma proteins and leukocytes to the site of infection

- alternative pathway provides a means of non-specific resistance against infection without the participation of antibodies and hence provides a first line of defense against a number of infectious agents.

2) Lecithin Pathway 

The lectin pathway of complement activation exploits the fact that many bacterial surfaces contain mannose sugar molecules in a characteristic spacing. The oligomeric plasma protein mannan-binding lectin (MBL; lectins are proteins binding sugars) binds to such a pattern of mannose moieties, activating proteases MASP-1 and MASP-2 (MASP=MBL activated serine protease, similar in structure to C1r and C1s). These, by cleaving C4 and C2, generate a second type of C3 convertase consisting of C4b and C2b, with ensuing events identical to those of the alternative pathway.

3) classical pathway

The classical pathway usually starts with antigen-bound antibodies recruiting the C1q component, followed by binding and sequential activation of C1r and C1s serine proteases. C1s cleaves C4 and C2, with C4b and C2b forming the C3 convertase of the classical pathway. Yet, this pathway can also be activated in the absence of antibodies by the plasma protein CRP (C-reactive protein), which binds to bacterial surfaces and is able to activate C1q.

Pharmacology cross reference: humanized monoclonal antibody Eculizumab binds to complement component C5, inhibiting its cleavage and preventing activation of the lytic pathway. This is desirable when unwanted complement activation causes hemolysis, as in paroxysmal nocturnal hemoglobinuria or in some forms of hemolytic uremic syndrome. For the lytic pathway's importance in fighting meningococcal infections, Eculizumab treatment increases the risk of these infections, which may be prevented by previous vaccination.

 BIOLOGICALLY ACTIVE PRODUCTS OF COMPLEMENT ACTIVATION

Activation of complement results in the production of several biologically active molecules which contribute to resistance, anaphylaxis and inflammation.

Kinin production
C2b generated during the classical pathway of C activation is a prokinin which becomes biologically active following enzymatic alteration by plasmin. Excess C2b production is prevented by limiting C2 activation by C1 inhibitor (C1-INH) also known as serpin which displaces C1rs from the C1qrs complex (Figure 10). A genetic deficiency of C1-INH results in an overproduction of C2b and is the cause of hereditary angioneurotic edema. This condition can be treated with Danazol which promotes C1-INH production or with ε-amino caproic acid which decreases plasmin activity.

Anaphylotoxins
C4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which cause basophil/mast cell degranulation and smooth muscle contraction. Undesirable effects of these peptides are controlled by carboxypeptidase B (C3a-INA).

Chemotactic Factors
C5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of neutrophils, basophils and macrophages and causes induction of adhesion molecules on vascular endothelial cells.

Opsonins
C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and promote phagocytosis.
Other Biologically active products of C activation
Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors and modulate their functions.

PHYSICAL AGENTS

Heat occupies the most important place as a physical agent.

Moist Heat : This is heating in the presence of water and can be employed in the following ways:

Temperature below 100°C: This includes holder method of Pasteurization where 60°C for 30 minutes is employed for sterilization and in its flash modification where in objects are subjected to a temperature of 71.1°C for 15 seconds. This method does not destroy spores.

Temperatures Around 100°C : Tyndallization is an example of this methodology in which steaming of the object is done for 30 minutes on each of three consecutive days. Spores which survive the heating process would germinate before the next thermal exposure and would then be killed.

Temperatures Above 100°C : Dry saturated steam acts as an excellent agent for sterilization. Autoclaves have been designed on the principles of moist heat.

Time-temperature relationship in heat sterilization
Moist heat   (autoclaving)

121°C       15 minutes
126°C         10 minutes
134 C          3 minutes

Dry heat

>160°C    >120 minutes
>170°C    >60minutes
>180°C    >30 minutes

Mechanism of microbial inactivation 

The autoclaving is in use for the sterilization of many ophthalmic and parentral products. surgical dressings, rubber gloves, bacteriological media as well a of lab and hospital reusable goods.

Dry Heat: Less efficient,  bacterial spores are most resistant. Spores may require a temperature of 140° C for three hours to get killed.
Dry heat sterilization is usually carried out by flaming as is done in microbiology laboratory to sterilize the inoculating loop and in hot air ovens in which a number of time-temperature combinations can be used. It is essential that hot air should circulate between the objects to be sterilized. Microbial inactivation by dry heat is primarily an oxidation process.

Dry heat is employed for sterilization of glassware glass syringes, oils and oily injections as well as metal instruments.    -

Indicators of Sterilization:  
These determine the efficacy of heat sterilization and can be in the form of spores of Bacillus stearothermophilus (killed at 121C in 12 minutes) or in the form of chemical indicators, autoclave tapes and thermocouples.

Ionizing Radiations

Ionizing radiations include X-rays, gamma rays and beta rays, and these induce defects in the microbial DNA synthesis is inhibited resulting in cell death. Spores are more resistant to ionizing radiations than nonsporulating bacteria.

The ionizing radiations are used for the sterilization of single use disposable medical items.

Mechanism of microbial inactivation by moist heat

Bacterial spores

•    Denaturation of  spore_epzymes
•    Impairment of germination
•    Damage to cell membrane
•    Increased sensitivity to inhibitory agents
•    Structural damage
•    Damage to chromosome

Nonsporulating bacteria

•    Damage to cytoplasmic membrane
•    Breakdown of RNA
•    Coagulation  of proteins
•    Damage to bacterial chromosome

Ultraviolet Radiations : 
wave length 240-280 nm have been found to be most efficient in sterilizing. Bacterial spores are more resistant to U.V. rays than the vegetative forms. Even viruses are sometimes more resistant than vegetative bacteria.

Mechanism of Action :

Exposure to UV rays results in the formation of purine and pyrimidine diamers between adjacent molecules in the same strand of DNA. This results into noncoding lesions in DNA and bacterial death.
Used to disinfect drinking water, obtaining pyrogen free water, air disinfection (especially in safety laboratories, hospitals, operation theatres) and in places where dangerous microorganisms are being handled.

Filteration

Type of Filters

Various types of filters that are available are    /
Unglazed ceramic filter (Chamberland and Doulton filters)
Asbestos filters (Seitz, Carlson and Sterimat filters)
Sintered glass filters

Membrane filters

Membrane filters are widely used now a days. Made up of cellulose ester and are most suitable for preparing_sterile solutions. The range of pore size in which these are available is 0.05-12 µm whereas the required pore size for sterlization is in range of 0.2-0.22 p.m.

Application of agglutination reactions

Agglutination reaction                Example

Tube agglutination    -> Widal test, Weil Felix reaction, Standard tube test for brucellosis

Slide agglutination   -> Typing of pneumococci,Diagnosis of Salmonella,Diagnosis of Shigella

Agglutination Absorption test  -> Salmonella diagnosis

Coagglutination   -> Grouping of streptococci, Identification of gonococci, Detection of Haemophilus, Antigen in CSF

Passive agglutination
Latex agglutination                   Detection of HBs Ag, ASO, CRP