OF BIOLOGICAL MATERIALS
From the 1993 Woodrow Wilson Biology Institute
Sterile technique is used to prevent bacterial, fungal and viral contamination. Contaminants generally fall into tubes or Petri dishes or are already present on utensils or in media. Therefore, sterile technique involves
- covering and then sterilizing utensils and media
- keeping contaminants out of the work area and
- keeping openings of tubes and Petri dishes covered
STERILIZATION OF UTENSILS AND MEDIA
Cover openings of flasks or beakers and wrap utensils, using aluminum foil. Heat in an autoclave or a pressure cooker (standard time: 15 minutes at 15 lbs psi pressure).
Autoclaves and pressure cookers operate at extremely high temperatures and under great pressure. Be certain that items have cooled sufficiently.
Flame-sterilize inoculation loops, scalpels and forceps by dipping in ethanol then passing through a flame to ignite the ethanol. Use the utensil when the flame goes out.
Containers of ethanol (used for dipping tools) and any object covered with ethanol will ignite readily. The flame is often difficult to see, so be certain that utensils are not burning before dipping into ethanol.
CREATING "STERILE" WORK AREAS
All work surfaces should be washed, then wiped with 70% ethanol.
Any movement around the work area can stir up air currents which can carry contaminants into the work area, despite other precautions. Attempt to minimize traffic around work areas.
A Bunsen burner provides an inexpensive and reasonable sterile field. The heat from the flame creates a convection current moving upwards around the flame. As a result, a small circumference around the burner is free of falling contaminants.
Work in an uncluttered area and keep loose clothing and hair away from flame.
Various (expensive) hoods provide an air-flow which pulls air and contaminants in one direction, usually toward the back of the hood. Working at the front (upstream) of the air current ensures that contaminants don't get pulled into media.
SAFEGUARDING THE OPENINGS
Tilt test tubes at an angle (about 45 degree) so that contaminants fall onto the side of the tube (instead of into the opening).
OF TUBES AND PETRI DISHES
Open Petri dishes as little as possible, holding the lid above the dish whenever possible.
Never place Petri-dish lids or test-tube caps onto a work surface. Find a way to hold onto the lids in an opening-down position. If placed face-up or face-down onto a surface, the lids can pick up contaminants that will later fall onto the media.
You can briefly flame the openings of flasks and test tubes before and after removing or adding anything to the containers. The heat will create a convection current and may kill some contaminants.
Place biological tissue into a 50 ml plastic centrifuge tube.
OF BIOLOGICAL MATERIALS
- Add approximately 25 ml of 70% ethanol to the tube (enough to cover the tissue). Replace the lid (snugly). Lay the tube on its side and rock it to constantly bathe the tissue in ethanol for 2-6 minutes.
- Carefully pour the ethanol into a waste beaker or a sink with running water. Be careful not to pour out the tissue. You may use sterile forceps to hold the tissue in the tube as you pour.
- Note: Hereafter, hold the test tube at an angle whenever the cap is not in place and use sterile techniques.
- Repeat step #2, adding 10% chlorine bleach instead of ethanol and bathe the tissue for 2-5 minutes. Watch the edges of the tissue for evidence of bleaching (the edges turn white). Pour off the bleach as soon as white edges appear or when 5 minutes have elapsed.
- 4. Repeat step #2 using sterile, distilled water instead of ethanol. Pour water off after 2-3 minutes. Perform this water rinse two more times.