BIOTECHNIQUES

1. BIOTECHNIQUES Concept of Sterilization Sterilization is the process carried out in biological experiments related to microbiology, animal & plant tissue culture, molecular biology, genetic engineering. Sterilization means complete destruction or removal of all the life forms specially micro-organisms from the given sample. If the material is not sterile then it is termed as contaminated.

2. Methods of sterilization… • 1. Physical • a. Filtration • b. Dry heat sterilization • c. wet heat sterilization • d. Radiation • 2. Chemical • a. Gaseous • b. Liquid sterilizers

3. Filtration: • It is the process of removing particulate matter from a liquid. • It is effective method of sterilization without heating. • Simple filters work by passing the solution through a filter with a pore diameter that is too small for microbes to pass through. • It removes but does not destroy microorganisms. • Generally two types of filters are used… • 1. Depth-filter • 2. Membrane filter

4. Membrane or Screen filters: • 1. Particulate filters: pore size ranges from 0.45 to 5 µm. • It removes particles of glass, plastic, rubber & other contaminants. • Special particulate filters are used to filter blood, emulsion, colloidal solutions. • 2. Microbial filters: pore size of 0.22 µm or smaller. • Ensures complete removal of microbes. • High Efficiency Particulate Air( HEPA) filters: used to deliver bacteria free air. • Used in laminar air flow.

5. Dry Heat Sterilization: • Dry heat sterilization used process like baking & flaming. • It does not use water or steam. • Protein hydrolysis can’t take place. • It kills the microbes by oxidation of cellular components. • This process requires more energy than protein hydrolysis & so higher temperatures are required for efficient sterilization by dry heat. • Objects are subjected to a temperature of at least 1600 C for 120 minutes.

6. Dry heat sterilization can be achieved by: • 1. Hot air sterilization • 2. Incineration • 1. Hot Air Sterilization: • Hot air sterilizer or oven is operated at higher temperature. • It is recommended where moist heat may produce undesirable changes in materials like oils, powders, wax & similar substances along with glassware like petri dishes. • For laboratory glassware a two hours exposure to a temperature of 1600 C is sufficient.

7. It was originally developed by Pasteur. • The oven uses dry heat to sterilize articles. • Generally they can be operated from 50 to 3000 C . • There is a thermostat for controlling the temperature. • Their double walled insulation keeps the heat in and conserves energy, the inner layer being poor conductor and outer layer being metallic. • Working of the hot air oven is based on the hot air inside the chamber of oven by the forced circulation. • As in any chamber hot air rises, so when the hot air reaches the top of chamber it is circulated to bottom by fan installed inside the chamber.

8. 2. Incineration: • Destruction by burning is known as incineration. • It is practiced routinely in the laboratory for sterilization of needles by using Busen burner. • During incineration care should be taken to prevent spattering, because droplets may carry viable organisms. • Incineration is also used for destruction of infected laboratory animals and other infected materials.

9. Wet heat Sterilization and Autoclaving: • Wet sterilization is also called as moist heat sterilization. • Moist heat can be used for destroying microorganisms by following ways… • (A) Steam under pressure: • Moist heat in the form of saturated steam is the most widely used and most dependable. • Steam sterilization is non-toxic, inexpensive, rapidly microbicidal, sporocidal. • Moist heat destroys microorganisms by irreversible denaturation of enzymes and structural proteins. • Laboratory apparatus used for steam under pressure is an autoclave.

10. Autoclave Autoclaves are of two types: Portable autoclave Stationary autoclave

11. Operation of Autoclave: • i) Articles to be sterilized are placed inside the apparatus. • Ii) Door is closed and clamped. • Iii) Heating is started & steam is generated. • Iv) Air vent is closed after complete removal of air. • V) when the pressure is reached at required 1210 C, sterilization time is timed. • Vi) At the end of the period, heat is disconnected. • Vii) Autoclave should not be opened before pressure is fallen down. • Air must be removed from autoclave.

12. Advantages of Autoclave: • 1. Rapid in operation as steam is stored in the walls (jackets). • 2. More economic as steam is trapped in the jacket and not allowed to escape into the atmosphere. • 3. material comes out dry as the inner surface is always warm.

13. B. Fractional Sterilization / Tyndalization: • Some media, solutions & biological materials cannot be heated above 1000 C, they are sterilized by this method. • Material is heated at 1000 C on three successive days with incubation periods between. • Resistant spores germinate during the incubation periods and converted to vegetative cells. • On subsequent heating vegetative cells will be destroyed.

14. Radiation sterilization: • The heat sensitive substances can be sterilized by this method. • Use of ionizing radiations to sterilize material is also known as cold sterilization. • They produce relatively little heat in material being killed. • Generally two types of radiations are used for sterilization… • Ionizing radiation • Non-ionizing radiation

15. Ionizing radiations: • These are short wavelength, high intensity radiations used to destroy microorganisms. • This radiation can come from gamma or X-rays that react with DNA resulting in damaging of the cell. 2. Non- ionizing radiations: • These are longer wavelengths & lower energy radiations. • They have less penetration ability. • Only uses for sterilizing surfaces. • The most common non-ionizing radiation is UV light.

16. Gamma rays: They are high energy radiations emitted from radioactive isotopes like Co60 . • They have great penetration power, so can be used for sterilization of materials of considerable thickness. • Examples: packaged foods & medical devices. (ii) X- rays: X-rays are lethal. They have considerable energy & penetration ability. • Still they are of little use for sterilization. • They are very expensive. • Difficult to utilize because radiations are given off in all directions.

17. (iii) Ultraviolet radiations( UV): it includes radiation from 150-3900A0 . • Many lamps are available which emit a high concentration of U.V. light (2600-2700). • Due to their poor penetration power, they are used only for surface sterilization. • Used in hospital operation theaters, pharmaceutical industry, food & dairy industry for treatment of contaminated surfaces. (iV) Cathode rays: cathode emits electrons, called cathode rays or electron beam. • When these elections are accelerated to extremely high velocities they can kill microorganisms. • Used in hospitals,(surgical appliances) drugs, packed materials.

18. Chemical Sterilization: • Due to application of heat, heat sensitive biological material or electronics or plastic will be damage therefore the chemicals either gases or liquid can be used as a sterilizer. • But sometime these chemicals may be harmful for human being. • Ethylene oxide and nitrogen dioxide are commonly used to sterilize the objects.

19. It is widely used by medical device manufacturer for large scale sterilization. • Recently ethylene oxide is classified as carcinogen. • Nitrogen dioxide gas is fast sterilants against variety of microorganisms. • Ozone: It is unstable and toxic gas therefore no practical use of such. In industrial level it is used to sterilize water and air. It has great oxidizing property to destroy wide range of pathogens.

20. Hydrogen peroxide: it is liquid and gas used as another sterilizer. It is strong oxidant and destroys many of the pathogens. It is used to sterilize heat sensitive devices like endoscopes. In medical it is used at higher concentration up to 90%. • Glutaraldehyde and Formaldehyde: these are the solutions generally used as fixatives but also used as sterilizers. For sterilization the object may be immersed in solution for longer period up to 24 hours. Formaldehyde is used in gaseous form. Vaccines may sterilized with formaldehyde. • Peracetic acid: The device like endoscopes is sterilized by standard peracetic acid.