WORKING WITH AGAR

A solid surface for bacterial growth is essential if organisms are to be isolated.  In the laboratories of Robert Koch, gelatin was first used to achieve isolated colonies.  Agar now serves as a more useful material.  It is a seaweed extract, which remains solid until heated to 100 degrees C (boiling).  Once liquefied, it can cool to 40-45 degrees C before resolidifying.  This gives adequate time and comfortable temperature range for pouring into the size and shape containers appropriate for a certain use.

You and your partner will be provided a bottle of melted agar and five sterile petri dishes.  The latter are shallow, round disposable plastic containers, which are pre-sterilized.  The top (lid) is larger and fits over the bottom dish.  Do not separate the top from the bottom except during the actual pouring.
 

  1. Begin with the dishes stacked. 
  2. Open the agar bottle.  (Be careful—it is still hot.  Handle it with three paper towels wrapped around it). 
  3. Heat the top of the bottle.  You may discard the lid.  Keep the bottle angled for pouring the rest of the procedure. 
  4. Holding the bottle with the left hand, move a Petri dish forward, raise the slightly with your right hand, pour the agar until the dish is 1/3 to ½ full, then lower it’s lid.
  5. Put the next dish on top of this one, lift it’s lid and pour. 
  6. Keep going until the bottle is empty and all dishes are up to ½ full.  You will end up with a stack of dishes.  The stacking allows them to cool more evenly and reduces condensation. 
  7. Discard the bottle according to the instructors directions.
  8. After one hour the now-solidified agar plates may be inverted.  All plates, sterile or inoculated, are stored inverted so that excess moisture absorbs back into the agar slowly rather than accumulating on the lid and dripping into the agar surface.

Avoiding environmental contamination during this procedure is essential.  Do not turn the bottle upright between pours since this would allow airborne particles to drop into the sterile agar.  Keep the lid of the dish being poured angled over its bottom to prevent the same airborne contamination.  Refrain from talking or excess movement in order to reduce air currents.

Label your plates on the edge with a grease pencil.  Put TSA or NA, and your initials.  These will be used next lab for environmental cultures and streak plate techniques.