Microbiology Guide: Introduction to Aseptic technique

Document ID

Document ID TE8820

Published Date

Published Date 07/27/2022
Microbiology Guide: Introduction to Aseptic technique
Microbiology Guide: Introduction to Aseptic technique.
From: Microbiology Guide: Media and Equipment Selection
  • Sterile work area for labs with cell culture hoods.
    • The simplest and most economical way to reduce contamination from airborne particles and aerosols (e.g., dust, spores, shed skin, sneezing) is to use a Class II or III cell culture hood.
    • The cell culture hood should be properly set up, and be located in an area that is free from drafts and traffic. The area should dedicated to the cell culture hood
    • The work surface should be uncluttered and contain only items required for a particular procedure; it should not be used as a storage area.
    • Before and after use, the work surface should be disinfected thoroughly, and the surrounding areas and equipment should be cleaned routinely.
    • For routine cleaning, wipe the work surface with 70% ethanol before and during work, especially after any spillage.
    • An ultraviolet light can be used to sterilize the air and work surfaces in the cell culture hood between uses.
    • Using a Bunsen burner for flaming is not necessary, nor is it recommended in a cell culture hood.
    • Leave the cell culture hood running at all times, turning it off only when it will not be used for extended periods of time.
  • Sterile Handling
    • Always wipe your hands and work area with 70% ethanol.
    • It is recommended to wear gloves. This will prevent any foreign contaminants from coming in contact with the customers and sample during testing. If gloves are not used, it is necessary to wash hands before and after testing.
    • Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell culture hood.
    • Avoid pouring media and reagents directly from bottles or flasks.
    • Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids, and use each pipette only once to avoid cross contamination.  Do not unwrap sterile pipettes until they are to be used.  Keep pipettes at the work area.
    • Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in resalable bags to prevent microorganisms and airborne contaminants from gaining entry.
    • Never uncover a sterile flask, bottle, Petri dish, etc. until the instant you are ready to use it and never leave it open to the environment.  Return the cover as soon as you are finished.
    • If you remove a cap or cover, and have to put it down on the work surface, place the cap with opening facing down.
    • Use only sterile glassware and other equipment.
    • Be careful not to talk, sing, or whistle while performing sterile procedures.
    • Perform experiments as rapidly as possible to minimize contamination.
  • Transferring bacteria from one sample to inoculate another sample.
    • Once a growing medium is poured into a Petri dish and cooled, it is referred to as a "plate." When a plate is inoculated, its surface is streaked with a tiny amount of a pure bacterial culture or a clinical sample. After inoculation, the plate is usually incubated for at least 24 hours to encourage growth of the sample. Time will vary dependent on method.
    • When working with Petri dishes, it is best to keep the lid of the dish closed, in order to avoid any particles in the air from landing on the plates. Mold can be a problem in humid areas. Mold appears as fuzzy growths on the agar (a picture is given below).
    • Bacterial inoculate can be transferred using an inoculation loop (a.k.a. inoculation wand). This instrument is essentially a wire with a small loop at one end and a handle at the other. The Loop, Inoculating, Wire (Product # 2112100) is made of metal and can be repeatedly used and then resterilized in a flame. After sterilizing the loop, it must cool briefly, so as not to kill organisms in the sample being transferred. When waiting for the loop to cool, do not wave it around to hasten cooling, and certainly don't blow on it. Either action could introduce bacterial contamination. If the customer states that he/ she hears a hissing sound when transferring sample by loop, it is because they have cooked and most likely killed any present  bacteria in there sample. Inoculating loops made of polystyrene are also available and are designed to be used only once before disposal. The Loop, Inoculating, Disposable, PS 10UL pk/25 (Product # 2749125) would be an example.
The following picture shows a plate that has been contaminated with mold.


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