ASEPTIC TECHNIQUE

 Laboratory practices that minimize the risk of cultured cells becoming contaminated with unwanted microorganisms

ASEPTIC TECHNIQUE HAS TWO GOALS:  Goal 1: Preventing contaminants from the environment from entering the culture  Goal 2: Preventing cultured cells from escaping to the environment where they could harm humans

KEY PRINCIPLES THAT GUIDE ASEPTIC TECHNIQUE  An object, surface, or solution is sterile if it contains no living organisms  Objects and solutions are considered non-sterile unless they have been treated to eliminate microorganisms  A sterile object, surface, or solution that is in contact with a nonsterile item is no longer sterile  Air is not sterile  Human skin and breath are rich sources of contaminating microorganisms

TOOLS TO MAINTAIN STERILE ENVIRONMENT  Autoclave  Laminar Flow Hood  Disinfectants  Bleach  Ethanol

AUTOCLAVE  An instrument that exposes substances to steam at elevated temperatures  In this class, we will use two types

WHY USE STEAM?  More penetrating than dry heat  Very effective at killing microorganisms  For sterilizing bacteria culture media, expose the media for 15 minutes at 121 o C at kPa

LAMINAR FLOW HOOD  Creates sterile air within the hood thereby, creating a sterile work surface  Air sterilized by flowing through a HEPA (high efficiency particulate air) filter  Removes particles 0.3  m or larger including most bacteria and microorganisms

LAMINAR FLOW HOOD  Horizontal laminar flow hood-useful for media preparation or sterile work with non-harmful organisms

THINK-PAIR-SHARE Explain why it is necessary to work in a laminar flow hood even though tools and solution have been sterilized in an autoclave? 3

SPECIFIC ASEPTIC PRACTICES FOR WORKING ON AN OPEN LAB BENCH  Expose materials and cultures to the environment for shortest period of time  Avoid contact between any sterile object and non-sterile item  Prepare a clean, clear area in which to work by swabbing surface with 70% alcohol

SPECIFIC ASEPTIC PRACTICES FOR WORKING ON AN OPEN LAB BENCH  Transfer bacteria from one place to another with inoculating loops sterilized by holding in a flame until they glow bright red  Don’t pass your arms, hands, or sleeves over open plate or flask to prevent contamination  Clean the work area and swab with 70% ethanol  Wash hands thoroughly