Sterilization after surgery

Aseptic Technique in the Lab: Complete Step-by-Step Guide & SOP (2025)

 

Ensuring aseptic conditions requires more than just sterilization of media, equipment, and laboratory environment. While these are prerequisites, personnel can carry various microorganisms that may recontaminate the environment. Combined with air circulation, this increases the risk of laboratory contamination. Therefore, mastering proper aseptic technique is crucial for successful experiments and must be strictly followed to ensure reliable results.

 

I. Definition of Aseptic Technique

A series of procedures and measures used in microbiological experiments to control or prevent contamination from various microorganisms. This encompasses two main aspects: creating a sterile operating environment and preventing the introduction of any foreign microorganisms during handling and cultivation.

 

II. Basic Principles

  1. 1. Distinguish between sterile and non-sterile zones; wear appropriate PPE (mask, gloves, and lab coat)
  2. 2. Only sterilized items should enter the biosafety cabinet; avoid overcrowding
  3. 3. Remove all unnecessary items after use and UV sterilize before shutting down
  4. 4. Perform operations near the Bunsen burner flame
  5. 5. Ensure inoculating loops are cooled before transferring target organisms
  6. 6. Flame-sterilize excess culture from inoculating loops to prevent environmental contamination

 

III. Aseptic Environment

The sterile room or clean room must be sanitized before use to create an aseptic working environment.

1. Clean Room Sterilization

  • - Clean thoroughly before use
  • - Wipe surfaces with sodium hypochlorite or benzalkonium chloride solution
  • - UV sterilize for minimum 30 minutes
  • - Ventilate for 10 minutes before entry
  • - Monthly formaldehyde fumigation with minimum two days ventilation
  • - Increase sterilization frequency as needed
  • - Regular sanitization for non-clean room laboratories
  • - Ozone treatment for labs working with E. coli phage contamination
  • - No unnecessary items allowed in sterile rooms

2. Biosafety Cabinet Preparation

  1. a) Pre-use: Wipe surfaces with 75% ethanol, place sterile materials inside, UV sterilize for 30 minutes
  2. b) During use: Run blower for 5 minutes before operations
  3. c) Post-use: Remove items, wipe surfaces, UV sterilize for 5 minutes, shut down

 

IV. Aseptic Techniques

  1. 1. Sterilizing Inoculating Loops Clean hands with 75% ethanol before entering the cabinet. Once ethanol has evaporated, light the Bunsen burner and heat the loop and shaft until red-hot, paying special attention to joints.
  2. 2. Cooling the Loop Allow sterilized loop to cool near the flame before use.
  3. 3. Inoculation Procedures Following media sterilization by autoclaving, transfer target organisms using sterile techniques within the biosafety cabinet.

a) Streak Plating

  • - Prepare cultures
  • - Sterilize loop
  • - Cool loop
  • - Sample culture: Sterilize glycerol stock tube with 75% ethanol, briefly flame, collect sample
  • - Streak plate: Flame plate rim, streak in sections with loop sterilization between each section
  • - Label plate with organism, date, and relevant information
  • - Incubate at appropriate temperature
  • - Store at 4°C after growth

b) Slant Inoculation

  • - Similar procedure but transfer single colonies to fresh slants using zigzag pattern
  • - Maintain sterile technique throughout
  • - Label and incubate appropriately

c) Liquid Culture Inoculation

Follow similar aseptic procedures as above.

 

V. Important Considerations

  1. 1. Maintain aseptic conditions; use proper PPE during cleaning and sterilization
  2. 2. Handle Bunsen burners safely to prevent fires
  3. 3. Clearly distinguish between sterile and non-sterile items
  4. 4. Remove unnecessary items from biosafety cabinet after use; properly sterilize contaminated materials
  5. 5. Regularly monitor lab contamination using settle plates
  6. 6. Minimize personnel movement during procedures
  7. 7. Avoid touching container openings to prevent contamination
  8. 8. In case of fire, remain calm and smother with damp cloth

 

These guidelines should be strictly followed to maintain aseptic conditions and ensure experimental success.