Heat Sterilization PDF

sterilization It is a process of complete destruction or removal of all living microorganisms within a system including bacteria, viruses, fungi, and spores which are resistant to most disinfectants and more resistant to heat than the non- sporing m...

sterilization It is a process of complete destruction or removal of all living microorganisms within a system including bacteria, viruses, fungi, and spores which are resistant to most disinfectants and more resistant to heat than the non- sporing microorganisms. It is critical in medical, laboratory, and food preparation settings to prevent infection, contamination, and ensure safety.  The process is all or none i.e. a system is sterile or non sterile and nothing in between, and the term sterile is therefore an absolute one. METHODS OF STERILIZATION ◆ 1- Physical methods: heat, UV, ionizing radiation and filtration. ◆ 2- Chemical methods: gas and liquid agents (sterilants). ◆ Sterilant: material or method used to remove or kill all microbes.  The effectiveness of any sterilization method depends upon: ◆ 1- Type of microorganism present: some are very difficult to kill, others die easily. ◆ 2- Number of microorganisms present: the less the number, the easier the sterilization process is. ◆ 3- Amount and type of organic material present: blood or tissue on poorly cleaned instruments act as a shield during the sterilization process. ◆ 4- Number of cracks and crevices on instruments: they collect and harbor microorganisms. ◆ 5- Choice of the right sterilization method. ◆ 6- Temperature. ◆ 7- Exposure time. ◆ 8- Concentration of the sterilizing material. ◆ 9- PH and environmental conditions. ◆ 10- Load configuration. ◆ 11- Validation and monitoring of the sterilization method. 1- Physical methods of sterilization A- Sterilization by heat 1- Sterilization by dry heat The heat is oxidation of microbial proteins. Sterilization with dry heat requires long exposure time and high temperature. ◆ The USP and BP specify the following temperature and time combinations for dry heat sterilization: ◆ 160°C (320°F) for 2 hours ◆ 170°C (338°F) for 1 hour ◆ 180°C (356°F) for 30 minutes ◆ These parameters are designed to ensure the effective killing of microbial life, including spores.  Techniques used for dry heat sterilization: 1- Direct flaming: Flaming and burning of platinum loop in microbiology, forceps in minor surgery. 2- Incineration: effective to sterilize disposable items and biological waste. ◆ 2- Hot air sterilization: Dry heat transfers heat less effectively to a cool body than moist heat. ◆ Standard conditions for dry heat sterilization: ◆ 1700c for one hour (total cycle time for placing instruments in oven, heating then cooling is from 2-2.5 hours), ◆ Or 1600c for two hours (total cycle time is from 3-3.5 hours). Key Features and Description of a Hot Air Oven: 1- Construction: - Outer Shell: Typically made of metal, the outer shell is insulated to maintain internal temperatures and prevent heat loss. - Inner Chamber: The interior is often made of stainless steel or other materials that can withstand high temperatures and are easy to clean. - Shelves/Racks: Adjustable shelves or racks are usually included to hold items being sterilized or dried, allowing for efficient use of space. 2- Heating Mechanism: - Heating Elements: The oven is equipped with electric heating elements that generate heat. These are usually located at the bottom or sides of the oven. - Temperature Control: A thermostat regulates the temperature within the chamber, ensuring consistent heating. 3- Air Circulation: - Forced Air Circulation: Many hot air ovens are equipped with a fan to circulate hot air evenly throughout the chamber, ensuring uniform temperature distribution and effective drying or sterilization. 4- Temperature Monitoring: - Thermometer: Internal or external temperature indicators monitor the chamber temperature. Some advanced models include digital displays for precise monitoring. - Calibration: Regular calibration is necessary to ensure accurate temperature readings and effective sterilization. 5- Door and Sealing: - Insulated Door: The oven features a tightly sealed door to prevent heat loss and maintain the desired internal temperature. The infra-red conveyor oven: Infra-red is a thermal radiation, i.e. when it is absorbed its energy is converted to heat. Therefore, it is often known simply as radiant heat. The infra-red conveyor oven makes maximum use of this radiant heat.  Infra-red oven consists of horizontal conveyor belt of wire mesh that moves through an insulated tunnel containing infra-red heating sources. The infra-red sources are concentrated at the entrance of the tunnel to heat the load quickly and the rest of infra-red sources are suitably spaced to maintain the temperature of the load. Temperature Control: Effective sterilization requires reaching and maintaining high temperatures for a specific duration. The infrared conveyor oven needs to be capable of reaching temperatures typically above 160°C (320°F) and maintaining them long enough to ensure sterilization. Uniform Exposure: The conveyor belt system ensures that items are exposed evenly to the infrared radiation, which helps achieve consistent sterilization across all surfaces.  Advantages Efficiency: The conveyor belt system allows for continuous operation and efficient processing of large quantities of items. Uniform Heating: Infrared radiation provides consistent heating, which helps in achieving uniform sterilization across all items. The infra-red vacuum oven: Long heating and cooling in air are undesirable for instruments as damage may occur. This is overcome by using infra-red vacuum oven which makes use of the fact that no carrier is required for heat transfer in infra-red sterilization and therefore it can be used in vacuum. The use of vacuum gives: 1- quicker heating up (as no heat is lost to the air) and 2- greater temperature stability (as there is no convection currents). 3- Vacuum also minimizes oxidation of metal instruments. ◆ The oven can reach and maintain the high temperatures needed to effectively kill microorganisms. The absence of air in the vacuum environment helps in achieving uniform heat distribution.  ALWAYS REMEMBER ✓ Exposure time begins only after the sterilizer has reached the target temperature. ✓ Do NOT overload the sterilizer. It alters heat convection and increases the time required for sterilization. ✓ Use sterile instruments immediately unless they are wrapped in paper or muslin, where they can be stored in a dry sterile container with a tight-fitting lid.  Application of dry heat sterilization: 1) Glassware e.g. flasks, beakers, tubes, containers, pipettes, and Petri dishes. They must be washed in hot water and detergent and well-rinsed. They are then dried in a drying oven at 65oC. 2) Porcelain and metal articles such as mortars, pestles and stainless steel dishes, scissors, scalpels and ointment tubes. 3) Oils and similar anhydrous materials: These materials (e.g. oint. bases as liquid, soft and hard paraffin, wool fat, wool alcohol, and bees wax, medical lubricants such as glycerol and vehicles used for oily injections such as fixed oils) are fluids at sterilization temperatures and convection will thus aid their heating up.  However, the preparation must be distributed in thin layers in small thin walled, shallow containers fitted with metal caps to heat up quickly. They are sterilized by dry heat to avoid contact with moisture which could deteriorate them.  When a sterile oily solution or suspension of a medicament is required to be prepared using one of those vehicles, the method of preparation depends on the thermostability of the medicament. For thermolabile medicaments such as procaine penicillin, the vehicle (arachis or sesame oil + 2% aluminum stearate) is sterilized separately, allowed to cool, and the medicament in then aspectically incorporated.  For thermostable drugs such as testosterone, progesterone, or estradiol benzoate, they are first incorporated into the oily vehicle and the completed preparation is then sterilized by dry heat. Therefore, they must be sterilized in small amounts or thin layers to avoid super-heating and damage of the outer layers of bulk powders. 4) Powders such as dusting powders (e.g. talc and kaolin) are also sterilized by dry heating to remain dry. The rate of heat transfer through them is very slow because of their low conductivity and the insulating effect of the air trapped between their particles. When the powder does not flow easily because its particles tend to stick together particularly if its moisture content is high (as in case of starch and sulfonamides) it must be dried at 100oC for about an hour, and then powdered before sterilization.  5) Paraffin gauze dressing is sterilized by dry heat at 150oC for 1 hour. It consists of a gauze of special paraffin. It is used in skin grafting and for treating burns and other wounds. The soft paraffin prevents adherence of the gauze to the tissues and the open nature of the gauze allows air to reach the wound and exudates to drain away into an outer layer of an absorbent dressing.  Advantage of dry heat sterilization: 1- Effectiveness Against Certain Microorganisms: o Effective for eliminating a wide range of bacteria, viruses, and fungi, including spores, when used correctly. 2- Non-Corrosive: o Unlike moist heat, dry heat does not corrode metal instruments, making it ideal for sterilizing tools and glassware. 3- No Residues: o Leaves no toxic residues, which is beneficial for materials that will come into contact with sensitive products. 4. Suitable for Certain Materials: o Ideal for sterilizing powders, oils, and non-aqueous solutions that could be damaged by moisture. o It is suitable for sterilization of assembled equipment. 5- Long Shelf Life: o Items sterilized by dry heat can be stored for extended periods without concerns of moisture- related degradation. 6- Simple Equipment: o Requires relatively simple and straightforward equipment (like a hot air oven), which can be cost- effective.  Disadvantage of dry heat sterilization: 1- Longer Sterilization Times: o Generally requires longer exposure times compared to moist heat methods. 2- Higher Temperatures Required: o Needs higher temperatures for effective sterilization, which may limit the types of materials that can be sterilized. 3- Poor Penetration: o Less effective in penetrating porous materials, making it unsuitable for certain instruments with intricate designs or internal structure, thus it is suitable for surface-only sterilization. 4- Risk of Damage: o High temperatures can potentially damage some materials if not monitored carefully. 5- Requires Proper Loading: o Needs careful loading of items in the oven to ensure proper heat circulation and uniform sterilization. 6- Ineffective for Heat-Sensitive Items: o Not suitable for sterilizing heat-sensitive materials, including certain plastics, wood and rubber. 7- Risk of Overheating: o There is a risk of overheating and potentially damaging heat-resistant items if not carefully monitored. o Therefore, it is unsuitable for sterilization of surgical dressings because the moisture of the fibers vaporizes quickly. This is followed by deterioration (discoloration and brittleness) and charring. 8- Energy Consumption o The high temperatures required can result in higher energy consumption, which might be less efficient compared to methods that use lower temperatures or shorter processing times.  2- Sterilization by Steam (Moist heat Sterilization) 1- Steam as a Sterilizing Agent: o Heat Transfer: Steam transfers heat more effectively than dry heat. When steam comes into contact with a cooler surface, it condenses, releasing latent heat. This heat transfer is critical for raising the temperature of the items being sterilized to the level required for effective microbial kill. ◆ Moisture Impact: The moisture from steam helps to denature proteins and disrupt cellular structures of microorganisms, leading to their destruction. This is particularly important for killing heat-resistant bacterial spores. o Any resistant protective layer of the microorganism can be softened by the steam allowing coagulation of its sensitive inner portions. o However, any greasy external material can hinder this effect, thus protecting the microorganism against the effect of steam. o This emphasizes the importance of thorough cleaning of objects prior to sterilization. 2- Temperature and Pressure Relationship: o Autoclaving: In most moist heat sterilization processes, such as autoclaving, steam is used at a pressure of 15-30 psi (pounds per square inch), which increases the boiling point of water to 121-134°C (250-273°F). This elevated temperature is necessary to achieve complete sterilization. o Pressure: The pressure in the autoclave allows steam to reach higher temperatures than boiling water alone, which is critical for achieving sterilization in a shorter time. 3- Sterilization Cycle: o Heating Phase: The items are exposed to steam at the required temperature for a specified period, depending on the temperature and type of material. o Exposure Time: The length of exposure time is critical to ensure that all microorganisms, including heat-resistant spores, are effectively killed. The exposure time is determined based on the temperature and the type of load being sterilized. o Cooling Phase: After the sterilization cycle, items are typically cooled under controlled conditions to avoid contamination. Proper drying may also be required to prevent moisture-related damage. 4- Penetration: o Uniform Distribution: Steam penetrates porous materials and complex items more effectively than dry heat. Proper steam penetration ensures that all surfaces and crevices of the items are exposed to the sterilizing conditions. 5- Microbial Inactivation: o Protein Denaturation: The high temperature and moisture from steam denature proteins within microorganisms, leading to their destruction. This process effectively inactivates bacterial cells, spores, and other pathogens. The greater resistance of microorganisms to dry heat is generally attributed to the greater heat stability of microbial proteins in the dry state. The temperature required to kill microorganisms is inversely related to the moisture present and so sterilization by moist heat could be done at lower temperatures than dry heat. This is because the mechanisms by which microorganisms are killed by moist heat is believed to be due to coagulation and denaturation of some proteins in the microbial cells. When proteins are heated in presence of water (i.e. wet proteins they release free SH groups and give rise to smaller peptide chains. These chains are mobile and can establish new bonds between themselves, thus forming new complexes different from the original protein molecules. In absence of water (dry heat), the protein molecules will be less active and so their mobility is much reduced.  Sterilization methods by moist heat: 1. Pasteurization: Pasteurization is a heat treatment process designed to kill or inactivate harmful microorganisms in liquids and certain foods without significantly affecting their taste or quality. This was introduced by Pasteur to prevent spoilage of wine by certain bacteria. In the process, the liquid or food is heated to a specific temperature for a set period of time and then rapidly cooled. The precise conditions depend on the type of pasteurization being used.  Types of Pasteurization: 1- Low-Temperature Long-Time (LTLT) Pasteurization: o Also known as batch pasteurization. It involves heating the liquid to 63°C (145°F) and holding it at this temperature for 30 minutes. 2- High-Temperature Short-Time (HTST) Pasteurization: o Also known as continuous pasteurization. It involves heating the liquid to 72°C (161°F) for at least 15 seconds. o This method is effective at reducing microbial load while preserving taste and nutrients. 3- Ultra-High Temperature (UHT) Pasteurization: o Also known as ultra-pasteurization. It involves heating the liquid to 135-150°C (275-302°F) for 2-5 seconds. o UHT treatment extends shelf life significantly without refrigeration. 4-Flash Pasteurization: o The liquid is typically heated to about 71.5°C (160°F) to 74°C (165°F) for about 15 to 30 seconds. The liquid is then quickly cooled to below 4°C (39°F) to prevent any further microbial growth. o This method is commonly used for pasteurizing beverages such as milk, fruit and juices. It effectively kills most pathogenic microorganisms without causing significant changes in taste or nutritional content. 5-Extended Shelf Life (ESL) Pasteurization: o A technique that combines pasteurization with additional processing steps to extend shelf life beyond that achieved by HTST. o It is often used for dairy products and juices to achieve longer shelf life while maintaining refrigeration requirements.  Advantages 1- Microbial Safety: Effective at reducing or eliminating pathogens like Salmonella, E. coli, and Listeria. It is useful to reduce the total bacterial count of milk and to kill its possible pathogens including: Rickettsia, Mycobacterium tuberculosis, Brucella and Streptococci. 2- Quality Preservation: Maintains the taste, texture, and nutritional value of the product better than some other methods of microbial control. 3- Shelf Life Extension: Improves the safety and shelf life of products.  Disadvantages 1- Not a Sterilization process: Does not achieve complete sterilization. Some microorganisms, especially heat-resistant spores, may survive the process. 2- Nutrient Loss: May cause some loss of vitamins and other sensitive nutrients, though generally minimal. 3- Limited to Specific Products: Primarily used for liquids like milk and juices and certain foods. Not suitable for all types of products. ◆ Process Considerations: Temperature and Time: The specific conditions depend on the pasteurization method and the product being treated. Cooling: Rapid cooling after heating is essential to prevent recontamination and maintain product quality.  2- Boiling: Description: Involves immersing items in boiling water to achieve sterilization. Process: Items are boiled in water at 100°C (212°F) for a period of time, usually 10-30 minutes. Uses: Commonly used for disinfecting, but not typically for complete sterilization as it may not effectively kill all bacterial spores. Often used for general cleaning and preparation.  3- Tyndallization Tyndallization, also known as fractional sterilization or intermittent sterilization, is a technique used to achieve sterilization through a series of heating cycles. It is used for heat-sensitive materials such as culture media, chemical solutions and biological materials, though less common today due to the availability of autoclaves. ◆ Tyndallization involves heating a substance to a temperature that kills most microorganisms, followed by incubation to allow any surviving spores to germinate into more heat-sensitive forms. The process is then repeated to kill the newly formed microorganisms. ◆ Process: – Heating: The material is heated to steam at 100°C (212°F) for a period of time, typically 30 minutes. – Cooling: After heating, the material is allowed to cool. – Incubation: The material is incubated at a temperature favorable for the growth of any surviving spores (e.g., 37°C or 98.6°F) to allow the spores to germinate. – Reheating: The material is then exposed to steam at 100°C for 30 minutes again. This cycle may be repeated for 2-3 days to ensure thorough sterilization. ◆ Advantages: 1- Effective for Heat-Sensitive Media: Tyndallization is suitable for sterilizing media or solutions that cannot withstand the higher temperatures and pressures of autoclaving (e.g., gelatin or some nutrient broths). 2- Eliminates Spore-Forming Microorganisms: The incubation periods between steam treatments allow heat-resistant bacterial spores to germinate into vegetative cells, which are then killed during subsequent steam exposures. 3- Preserves Nutrient Integrity: It minimizes damage to sensitive nutrients in culture media, ensuring their functionality post- sterilization. 4- No Special Equipment Required: Unlike autoclaving, tyndallization does not require pressurized equipment. It can be carried out using a simple steamer or boiling setup. ◆ Disadvantages: 1- Time-Consuming: The process is relatively lengthy, as it requires multiple heating and cooling cycles over several days. 2- Not as Reliable as Modern Methods: While effective for some applications, Tyndallization is less reliable compared to modern methods like autoclaving, which provides more consistent and complete sterilization. 3- Not Suitable for All Substances: It is ineffective for sterilizing materials that cannot be exposed to prolonged steam or for substances with heat-stable spores that might survive the process. 4- Limited Penetration: It may not be suitable for penetrating porous materials effectively.  4- Heating with a bactericide: ◆ Heating with a bactericide involves combining heat with a chemical agent designed to kill or inhibit the growth of bacteria. This approach can be used to enhance the effectiveness of sterilization or disinfection processes. ◆ Sterilization by heating with a bactericide is often used in situations where lower temperatures are needed to protect the integrity of the material being sterilized. ◆ Common bactericides like phenol, formaldehyde, alcohols, and hydrogen peroxide can be combined with heat to improve sterilization efficacy, particularly for heat- sensitive biological materials.  Common Bactericides in Heat Sterilization of Drugs and Biologicals: 1. Formaldehyde Formaldehyde is used in the sterilization of certain biological products such as vaccines and therapeutic proteins. 2. Phenolic Compounds Phenolic bactericides like cresols, chlorocresols or chloroxylenol are occasionally used in the heat sterilization of pharmaceutical products, particularly those that are stable in the presence of these chemicals. 3- Chlorhexidine Chlorhexidine is used as an antiseptic and bactericide in some drug formulations, particularly in ophthalmic solutions, mouthwashes, and topical preparations. It is also used to sterilize medical equipment in combination with heat. 4-Thiomersal Thiomersal, a mercury-based preservative, is used in certain vaccines and biological products to prevent bacterial and fungal contamination. The use of bactericides in intravenous (IV) injections is generally not recommended and is subject to strict guidelines due to safety concerns. Most bactericides are not suitable for IV solutions, but some preservatives may be used in specific situations and in low concentration (e.g., multi-dose vials). They can cause irritation or adverse reactions when injected. ◆ Exceptions Benzyl Alcohol: Occasionally used as a bacteriostatic agent in multi-dose vials, but it is contraindicated in neonates due to toxicity. In adults, it should be used with caution and only in specified concentrations. Methylparaben and Propylparaben: These may be used in very low concentrations in some formulations but are not appropriate for large- volume or continuous infusions due to potential irritant effects. The use of bactericides in intrathecal injections is highly discouraged. The intrathecal space is sensitive, and any additive, including bactericides, can cause severe irritation, inflammation, or even neurotoxicity.  5- Moist heat at temperature above 100oC (autoclaving): *Autoclaving is the most reliable method and is the most widely used for sterilization. *It is highly effective at killing all types of microorganisms, including bacteria, viruses, fungi, and bacterial spores. * Articles to be sterilized are subjected to saturated steam at a temperature higher than 100oC. i.e. subjected to steam under pressure. The principle employed in autoclaving to get steam under pressure depends on the fact that water boils when its vapor pressure equals the pressure of the surrounding atmosphere. Thus, boiling occurs at 100oC at normal atmospheric pressure.  However, when water is boiled in a closed vessel, the pressure will increase and hence the temperature at which water boils will consequently increase. The temperature of the steam generated from the water boiling under pressure will then rise above 100oC.  The apparatus for sterilization by steam under pressure is called an “autoclave” and the official conditions for autoclaving are 115- 116oC for 30 minutes (B.P.) 121oC for 15 minutes (U.S.P.) excluding heating-up times.  Stationary autoclaves ◆ Also known as conventional autoclaves. They are large, fixed sterilization devices designed to use steam under pressure to sterilize equipment, tools, and materials in various settings.  Portable autoclave: Steam is produced inside the autoclave and it is usually wet because it is in constant contact with boiling water. The simple type of portable autoclave consists of a vertical or horizontal cylinder of metal or stainless steel of about 40 cm in diameter and 65 cm in length. The cylinder has a lid fastened by screw clamps and having a rubber gasket to render it air tight. There are three controls on the upper side of the lid: 1-a vent (discharge tap through which air is expelled), 2- a pressure gauge to indicate the pressure inside the autoclave, 3-and a safety valve to allow the passage of excess steam. 4- Temperature sensors are placed inside the autoclave chamber and are designed to monitor and regulate the temperature during each cycle. The cylinder contains water up to a certain level (about 1/5 of the internal height) which is heated by gas or electricity below the cylinder. The articles to be sterilized are loosely arranged on a perforated tray situated above the water level. Tight packing is avoided to leave space for expansion and prevent breakages and also to allow air to escape easily.  Sterilization cycle ◆ 1. Pre-Sterilization Phase ◆ a. Loading: Materials to be sterilized are placed inside the autoclave. Care is taken to ensure proper spacing for steam circulation and to avoid overloading, which could compromise sterilization. For liquids, containers should not be sealed tightly to allow pressure equalization. ◆ b. Air Removal: Effective sterilization requires the removal of air (since air pockets can insulate microorganisms). Depending on the autoclave type: Gravity Displacement Autoclave: Steam forces air out of the chamber through a vent. Vacuum Autoclave: Pre-vacuum actively remove air for more efficient steam penetration. ◆ 2. Sterilization Phase Once air is removed, the chamber reaches the sterilization conditions: Temperature: Commonly 121°C or 134°C, depending on the load type. Pressure: 15 psi (103 kPa) for 121°C, or 30 psi (207 kPa) for 134°C.  The sterilization temperature is maintained for a specified duration: Standard Times: 121°C for 15–20 minutes (used for most loads). 134°C for 3–5 minutes (used for flash sterilization). This phase kills microorganisms, including bacterial spores, by denaturing proteins and disrupting cellular structures. ◆ 3. Depressurization Phase After sterilization, the chamber pressure is gradually reduced to atmospheric pressure. For liquids, this is done slowly to prevent damage to containers. ◆ 4. Cooling and Drying Phase Non-Liquid Loads: A drying phase may follow, where the chamber is evacuated to remove moisture from the sterilized items. Liquid Loads: Liquid sterilization cycles do not include a drying phase, as the containers are typically sealed immediately after cooling. ◆ 5. Unloading Phase Sterilized materials are removed from the autoclave. Items should be handled with sterile gloves to avoid contamination and left to cool to room temperature in a sterile area if necessary.  Advantages of moist heat sterilization: High Efficacy: Effective at killing all types of microorganisms, including spores. Versatility: Suitable for a wide range of materials and items. Reliability: Provides consistent and reliable sterilization results. Safety: low toxicity and no residue. Lower temperature requirement. Enhanced penetration. Cost-effectiveness.  Limitations: ◆ Moisture Sensitivity: Materials that are damaged by heat and moisture cannot be autoclaved. ◆ Size Limitations: Large items or complex equipment may not fit into smaller autoclaves. ◆ Biofilm and Soil Residue: Any organic matter (biofilms or blood) left on instruments can shield microbes from steam, rendering the process ineffective. ◆ Corrosion risk: for metal instruments. ◆ Maintenance and Calibration: Regular calibration and cleaning are essential for ensuring the autoclave operates effectively and consistently. ◆ Long cycle time.  Hydrostatic continuous sterilizers: ◆ Autoclaves are not ideal for sterilizing large industrial batches of intravenous fluids because of their low capacity as well as large staff and space requirements. ◆ Inaddition, bacterial multiplication and pyrogen production may occur in bottles that wait autoclaving. ◆ Hence, some pharmaceutical companies use continuous sterilizers capable of handling up to 12000 or more bottles per hour. ◆ N.B.: Pyrogen: fever producing substance. ◆ Hydrostatic continuous sterilizers are specialized equipment used in pharmaceutical and food industries for sterilizing products continuously without interrupting production. ◆ They are especially valuable in large-scale operations that require consistent sterilization of liquid or semi-liquid products like parenteral solutions, or heat- stable biologicals.  Air Removal from autoclaves: The presence of even small proportion of air can have considerable effects on the properties of the steam. The effect of air on steam is two-fold:  Superheating: Pure saturated steam has a definite temperature at a particular pressure. The temperature of a mixture of air and steam will be lower than the temperature of saturated steam at the same pressure. The temperature of the mixture will depend on the proportion of air because air gives much smaller heat than steam of equivalent pressure.  Consequently, the reading of the gauge pressure of an autoclave cannot be converted to the equivalent saturated steam temperature unless all the air is expelled. In the presence of air, if the thermometer of the autoclave show a temperature corresponding to saturated steam at the indicated pressure this means the steam is superheated.  Obstruction or delay of heat penetration: ◆ Air from an air/steam mixture reduces the heat transfer and prevents penetration of steam and renders some materials unsterilizable. ◆ Without proper condensation and heat transfer, microbial cells may not be destroyed completely. This can lead to sterilization failures and potential contamination risks.  Causes of superheating: 1- Air trapping or inadequate air removal. 2- Insufficient steam generation. 3- Improper load configuration. 4- Temperature inconsistencies. 5- Pressure-Temperature Imbalances. ◆ Preventing Superheating 1- Ensure Steam Saturation 2- Proper Chamber Air Venting 3- Load Items Properly 4- Monitor Pressure and Temperature  Mechanism of thermal destruction Of the two methods used, the dry heat and moist heat, thermal death of microorganisms takes place as the result of inactivation of essential cellular proteins or enzymes. But one should differentiate between dry and moist heat. ◆ During dry heat and moist heat, thermal death of microorganisms takes place as the result of inactivation of essential cellular proteins or enzymes. ◆ The greater resistance to dry heat is generally attributed to heat stability of proteins in the dry heat.  Death by dry heat is primarily an oxidation process while death by moist heat is due to coagulation of some proteins in the cell and that oxidation goes more slowly than coagulation. ◆ When wet proteins are heated they release free SH-groups and give rise to smaller peptide chains. ◆ These chains are mobile and show the capacity to form negative bonds between themselves, thus forming new complexes different from the original protein molecules. ◆ In the absence of water, the polar groups in the peptide chains are less active, so that their mobility is much reduced. ◆ Therefore, it requires more energy to open the peptide molecules, hence the increased apparent resistance of the protein in the dry state. Kinetics of thermal Destruction: ◆ Expression of resistance ◆ The thermal death point (TDP) is the lowest temperature at which all microorganisms in a liquid suspension or culture are killed within a specific time. ◆ It is a critical parameter in microbiology and sterilization processes, used to determine the effectiveness of heat treatments in eliminating microorganisms. ◆ Thermal death Time (TDT): The TDT is the time required to kill a specific microorganism at a given temperature. It is crucial for ensuring that the conditions used in sterilization or pasteurization processes are sufficient to achieve complete microbial inactivation. It is affected by many factors including the type of microorganism, temperature, medium and conditions, and heat transfer efficiency. ◆ D- value ◆ This value measures the degree of resistance of an organism to a sterilizing agent ◆ It refers to Decimal Reduction Time (DRT). ◆ It is the time taken at a fixed temperature to reduce the number of viable organism by 90%. ◆ Z-value: ◆ Measure the sensitivity in terms of different temperatures. ◆ Z-value is the increase in temperature needed to reduce the D-value at any temperature by 90%. ◆ The more resistant an organism, the larger will be the Z-value and vice versa.