Shreffler:Aseptic Technique: Difference between revisions

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*Put 3 mL of LB medium into a 15 mL tube
* Protect eyes, mucous membranes, open cuts and wounds from contact with biohazardous material.
#Add 1.5 μL carbinicillin (final concentration 50 μg/mL)
* Use personal protective equipment as necessary (i.e. gloves, goggles, mask, other protective clothing).
#With a sterile toothpick held by flamed tweezers, pick a colony off the plate and place the toothpick into the 3 mL of medium
* Carry out all culture operations under a laminar flow hood.
#Place in shaker overnight (approximately 16 hours) at 37°C at a speed of 240 rpm
* Disinfect all surfaces with a disinfectant solution prior to use.
#Carefully remove and add 75 μL of carbinicillin to the two 150 mL flasks of LB medium previously prepared
** Swab down the working surface liberally with 70% ethanol solution.
#For the next overnight culture, add 300 μL of the first overnight culture to each of the flasks (1 to 500 dilution)
** Periodically spread 70% ethanol solution over the exterior of gloves to minimize contamination.
#Place in shaker overnight (approximately 16 hours) at 37°C  at 240 rpm
** Swab all glassware (medium bottles, beakers, etc.) with 70% ethanol solution before placing them inside the hood.
** In case of any spill, spread 70% ethanol solution and swab immediately with non-linting wipes.
* Replace gloves if torn, and discard after use. Do not wear them when entering any other lab area.
* Bring into the work area only those items needed for a particular procedure.
** Leave a wide clear space in the center of the hood (not just the front edge) to work on.
** To prevent blockage of proper air flow and to minimize turbulence, do not clutter the work area.
** Make sure to have easy access to all items. Do not reach over an item to retrieve another (especially over an open bottle or flask).
* Never perform mouth pipetting; always use a pipetter.
** Use sterile wrapped pipettes and discard them into a biohazard waste container after use. Check that the pipette wrapping is not broken or damaged, to confirm sterility.
** Make sure not to touch the tip of the pipette to the rim of any flask or sterile bottle.
* Inspect the vessels to be used:
** T-flask - Must be free of visible contamination or breakage, or lack container identification. The plastic covering the flask must be intact.
** Bottles - Check for cracks and expiration dates.
** Spinner flasks - Check for cracks, expiration dates, and proper assembly.
* When handling sterile containers with caps or lids, place the cap on its side if it must be laid on the work surface.
* Discard any biohazardous or contaminated material immediately.
* When finished, clean the work area by wiping with 70% ethanol solution.




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[[Category:ShreffLab]]
[[Category:ShreffLab]]
Aseptic Technique
• Protect eyes, mucous membranes, open cuts and wounds from contact with biohazard material.
• Use gloves, goggles, mask, and protective clothing as necessary.
• Carry out all culturing operation is a laminar flow hood.
• Disinfect all surfaces prior to use with a disinfectant solution.
• Swab down the working surface liberally with 70% ethanol..
• Periodically spread a solution of 70% ethanol over the exterior of gloves to minimize contamination. Replace them if torn.
• In case of any spill, spread a solution of 70% alcohol and swab immediately with non-linting wipes.
• Discard gloves after use and do not wear them when entering any other lab area. Bring into the work area only those items needed for a particular procedure.
• Leave a wide clear space in the center of the hood (not just the front edge) to work on. Do not clutter the area to prevent blockage of proper air flow and to minimize turbulence.
• Swab with 70% alcohol all glassware (medium bottles, beakers, etc.) before placing them inside
• the hood.
• Arrange the work area to have easy access to all of it without having to reach over one item to get at another (especially over an open bottle or flask).
• Use sterile wrapped pipets and discard them after use into a biohazard waste container.
• Check that the wrapping of the sterile pipet is not broken or damaged.
• Inspect the vessels to be used:
• T-flask - Must be free from visible contamination or breakage, or lack container identification. The plastic covering the flask must be intact.
• Bottles - Check for cracks, expiration dates.
• Spinner flasks - Check for cracks, expiration dates, and proper assembly.
• Discard any biohazard or contaminated material immediately..
• Never perform mouth pipetting. Pipettor must be used.
• When handling sterile containers with caps or lids, place the cap on its side if it must be laid on the work surface.
• Make sure not to touch the tip of the pipette to the rim of any flask or sterile bottle.
• Clean the work area when finished by wiping with 70% alcohol. 18.

Latest revision as of 13:05, 3 December 2009

General Guidelines

  • Protect eyes, mucous membranes, open cuts and wounds from contact with biohazardous material.
  • Use personal protective equipment as necessary (i.e. gloves, goggles, mask, other protective clothing).
  • Carry out all culture operations under a laminar flow hood.
  • Disinfect all surfaces with a disinfectant solution prior to use.
    • Swab down the working surface liberally with 70% ethanol solution.
    • Periodically spread 70% ethanol solution over the exterior of gloves to minimize contamination.
    • Swab all glassware (medium bottles, beakers, etc.) with 70% ethanol solution before placing them inside the hood.
    • In case of any spill, spread 70% ethanol solution and swab immediately with non-linting wipes.
  • Replace gloves if torn, and discard after use. Do not wear them when entering any other lab area.
  • Bring into the work area only those items needed for a particular procedure.
    • Leave a wide clear space in the center of the hood (not just the front edge) to work on.
    • To prevent blockage of proper air flow and to minimize turbulence, do not clutter the work area.
    • Make sure to have easy access to all items. Do not reach over an item to retrieve another (especially over an open bottle or flask).
  • Never perform mouth pipetting; always use a pipetter.
    • Use sterile wrapped pipettes and discard them into a biohazard waste container after use. Check that the pipette wrapping is not broken or damaged, to confirm sterility.
    • Make sure not to touch the tip of the pipette to the rim of any flask or sterile bottle.
  • Inspect the vessels to be used:
    • T-flask - Must be free of visible contamination or breakage, or lack container identification. The plastic covering the flask must be intact.
    • Bottles - Check for cracks and expiration dates.
    • Spinner flasks - Check for cracks, expiration dates, and proper assembly.
  • When handling sterile containers with caps or lids, place the cap on its side if it must be laid on the work surface.
  • Discard any biohazardous or contaminated material immediately.
  • When finished, clean the work area by wiping with 70% ethanol solution.


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