Sterilization and Disinfection
Safety in the Laboratory
Pathogenic organisms present in specimens require strict safety protocols.
Microbiology labs have heightened risk and must work under biological safety conditions.
Risk Groups
Risk Group 1: Low individual/community risk (e.g., E. coli K12, Lactobacilli).
Risk Group 2: Moderate individual risk/low community risk (e.g., Staphylococcus, Salmonella species).
Risk Group 3: High individual risk/low community risk (e.g., M. tuberculosis).
Risk Group 4: High individual/community risk (e.g., Ebola virus).
Antiseptic: process used to decrease the number of microorganism that are present on skin.
Disinfection vs. Sterilization
Disinfection: Destroys pathogenic organisms; does not guarantee spore destruction.
Methods: Physical (heat) and chemical (bleach-1/10 water, alcohol-70% more effective)
Sterilization: Kills or destroys all microorganisms, including spores.
Methods: Physical (heat, filtration) and chemical means.
Sterilization by Dry Heat involves exposing materials to high temperatures (typically 160-180°C) for a specific duration to ensure the destruction of all forms of microbial life, including resistant spores.
Sterilization by moist heat, commonly achieved through autoclaving, utilizes steam at high pressure (typically 121°C or higher) to effectively kill microorganisms and spores in a shorter time frame compared to dry heat. Chemical sterilization involves the use of gaseous agents, such as ethylene oxide or hydrogen peroxide, which can penetrate materials and eliminate all forms of microbial life, often used for items that cannot withstand high temperatures.
Autoclave Operation
Uses steam under pressure to achieve high temperatures for sterilization.
The material is placed in a special autoclave bag or container.
Required conditions: 121°C, 103.4 kpa (15 psi) for 15-60 minutes.
Steps to operate: clean, load items correctly, monitor parameters, and document cycles.
Autoclaving Procedures
Use plastic only if it's polypropylene; avoid sealed containers.
Follow loading rules for proper steam penetration and prevent crowding.
Items that CAN be autoclaved include metal instruments, glassware, and certain types of durable rubber, ensuring that they are free of debris and moisture before the process.
Items that CANNOT be autoclaved are materials containing: solvents, volatile, chlorinated compounds (HCL, bleach) or corrosive chemicals (phenol, trichloroacetic acid, ether, chloroform) certain types of plastics like polyvinyl chloride (PVC), and any items that may be damaged by heat or moisture, such as electrical equipment and delicate instruments.
Indicators of Sterilization
Chemical Indicators: Change color to indicate proper temperature/time.
Biological Indicators: Use heat-resistant spores to verify effectiveness.
Disadvantages of biological indicators: they require specific conditions for growth, which may lead to delays in obtaining results, and may also be more costly compared to chemical indicators. Additionally, their interpretation can be complex, as they necessitate careful analysis to confirm sterilization, whereas chemical indicators provide immediate visual feedback.
Biological indicators contain spores of a heat-resistant bacterium Geobacillus stearothermophilus, which is commonly used due to its resilience to high temperatures and its effectiveness in challenging the sterilization process.
Autoclave Maintenance
Daily: Disinfect surfaces, check gaskets, clean interiors.
Yearly: Professional inspection; maintain documentation for compliance.
Fundamental Rules
Place empty flasks, test tubes, or other non-porous containers on their side with loose covers. This provides horizontal airflow that helps to minimize contamination during the sterilization process.
Common Hazards
Risks of heat/steam burns, glass breakage, chemical reactions from autoclaving.
Troubleshooting Sterilization Issues
Common problems: inadequate temperature, cycle time, or steam penetration.