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.
 

NITRIC OXIDE-DEPENDENT KILLING

Binding of bacteria to macrophages, particularly binding via Toll-like receptors, results in the production of TNF-alpha, which acts in an autocrine manner to induce the expression of the inducible nitric oxide synthetase gene (i-nos ) resulting in the production of nitric oxide (NO) . If the cell is also exposed to interferon gamma (IFN-gamma) additional nitric oxide will be produced (figure 12). Nitric oxide released by the cell is toxic and can kill microorganism in the vicinity of the macrophage.

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.

Immunology:

The branch of life science which deals with immune reaction is known as immunology.

Components of Immune System:

The immune system consists of a network of diverse organs and tissue which vary structurally as well as functionally from each other. These organs remain spreaded throughout the body. Basically, immune system is a complex network of lymphoid organs, tissues and cells.

These lym­phoid organs can be categorized under three types depending upon their functional aspects:

i.  Primary lymphoid organ.

ii. Secondary lymphoid organ.

iii.Tertiary lymphoid organ.

White blood cells or leukocytes are the basic cell types which help to give rise to different types of cells which participate in the development of immune response . WBC are classified into granulocytes and agranulocytes depending on the presence or absence of granules in the cyto­plasm.

Agranular leukocytes are of two types, viz., lymphocytes and monocytes. Lymphocytes play pivotal role in producing defensive molecules of immune system. Out of all leukocytes, only lymphocytes possess the quality of diversity, specificity, memory and self-non self recognition as various important aspects of immune response.

Other cell types remain as accessory one; help to activate lymphocytes, to generate various immune effector cells, to increase the rate of anti­gen clearance 

All cells of the immune system have their origin in the bone marrow 

myeloid (neutrophils, basophils, eosinpophils, macrophages and dendritic cells) 

lymphoid (B lymphocyte, T lymphocyte and Natural Killer) cells .

The myeloid progenitor (stem) cell in the bone marrow gives rise to erythrocytes, platelets, neutrophils, monocytes/macrophages and dendritic cells whereas the lymphoid progenitor (stem) cell gives rise to the NK, T cells and B cells. 

For T cell development the precursor T cells must migrate to the thymus where they undergo differentiation into two distinct types of T cells, the CD4+ T helper cell and the CD8+ pre-cytotoxic T cell. 

Two types of T helper cells are produced in the thymus the TH1 cells, which help the CD8+ pre-cytotoxic cells to differentiate into cytotoxic T cells, and TH2 cells, which help B cells, differentiate into plasma cells, which secrete antibodies. 

Function of the immune system is self/non-self discrimination. 

This ability to distinguish between self and non-self is necessary to protect the organism from invading pathogens and to eliminate modified or altered cells (e.g. malignant cells). 

Since pathogens may replicate intracellularly (viruses and some bacteria and parasites) or extracellularly (most bacteria, fungi and parasites), different components of the immune system have evolved to protect against these different types of pathogens.

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.