Microbiology Guide: Introduction to Aseptic technique

Document ID

Document ID TE8820

Published Date

Published Date 06/06/2018
Question
Microbiology Guide: Introduction to Aseptic technique
Summary
Microbiology Guide: Introduction to Aseptic technique.
Answer
From: Microbiology Guide: Media and Equipment Selection

It is recommended to wear gloves. This will prevent any foreign containments 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.
  • Optimal situations: the use of a hood. Most of our customers may not have this available so here are a few tips to best avoid cross contamination of samples by keeping a clean environment.
  • When working with Petri dishes it is best to keep the lid of dish closed to avoid any particles in the air to land on the plates. Hach agar is pretty specific but on occasion “Fuzzies” might appear on plates, his is mainly mold and very common in humid areas (i.e. Florida).
  • Sterile work area for culture hooded labs.
    • 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 cell culture hood. 
    • The cell culture hood should be properly set up and be located in an area that is restricted to cell culture that is free from drafts from doors, windows, and other equipment, and with no through traffic. 
    • 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 exposed 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 they will not be used for extended periods of time.
  • Sterile Handling
    • Always wipe your hands and your work area with 70% ethanol.
    • 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 your pipettes at your 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 when you are performing sterile procedures. 
    • Perform your 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 a “plate.” Plates are made ultimately to be inoculated, that is, have a tiny amount of a pure bacterial culture or a clinical sample streaked across the surface. After inoculation, the plate is usually incubated for at least 24 hours to encourage growth of the sample. Time will vary dependent on method.
    • 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. Since it is made of metal, it 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. Plastic loops are also available but are designed to be only used once and then disposed of.

Options:
Reusable Wire Inoculating Loop (Catalog number/Order code 2112100)
Disposable Inoculating Loop pack of 25 (Catalog number/Order code 2749125)


 
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