Revision (original) 4/2020

Flame sterilization is a standard microbiology technique. If possible, disposable sterile inoculating loops and cell spreaders should be substituted for flame sterilization to eliminate the need for open flame. If this technique must be used, researchers must ensure that the ethanol and other sources of combustion are not too close to the open flame, and that the open flame is positioned in a place which limits the passage of your hands near the fire. Researchers must adhere to the following steps when flame sterilizing on the open bench.

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PPE and Attire

PPE and Attire

Personal protective equipment, including a lab coat, gloves, and safety glasses MUST BE WORN. Tie back long hair and remove dangling jewelry. Appropriate lab attire, including long pants and close toed shoes are required in all Penn labs.

Work Area Preparation

Work Area Preparation

Clean your work bench and remove all unnecessary items from the immediate work area, especially flammable items and chemicals. Ensure that no combustible materials are on shelves above or below the work area.  Note the location of the nearest eye wash and emergency shower.

Ethanol Preparation

Ethanol Preparation

Pour ethanol into a secondary container made of PYREX GLASS or METAL, wide enough to dip a spreader or inoculating loop. Check the integrity of glass containers to ensure they are free of cracks or chips.  The container should have an easily replaceable lid to smother ignited ethanol. Only a small volume of ethanol is needed, enough to cover the end of the loop or spreader (approximately 1 cm or less in depth or about 10 ml in volume). An ideal container is a Pyrex Glass Petri Dish. Clean up any ethanol that may have spilled while pouring from the stock bottle into the container before beginning work.

Workflow

Workflow

Review and organize the work area before each use.

The ethanol container should be kept as far away as is reasonable from the flame and should be positioned in the work area so that it is not necessary to pass the flamed sterile instrument back over the ethanol container prior to immediate use. Ensure all ethanol is burned off before moving the instrument to back over the main work area.

Liquid cultures and petri dishes should be kept reasonably close to the flame to maintain a sterile environment by utilizing the updraft of air created by the flame.

Diagram showing position of materials on benchtop
This is an example of a bench set-up for someone who pipets with the RIGHT HAND. It is best to keep the flame near the hand you use less, as this will decrease the likelihood of your working hand passing too close to the flame.

 

Emergency Response

Emergency Response

If the ethanol ignites a fire that cannot be smothered, evacuate the area while notifying others of the fire.

Do not use a fire extinguishers to put out the fire unless you have been trained by Penn EHRS and FES. Upon leaving the lab, shut the door and pull the fire alarm. Once outside of the building, report to one of the designated Building Areas of Refuge and call PennComm (215-573-3333) to report the fire (Philadelphia Campus).

General fire response information and emergency contact numbers for other Penn campuses can be found at Emergency Info.