XIX.  ASEPTIC TECHNIQUE


  Introduction

  This module will introduce you to methods used in culturing microorganisms (e.g., bacteria, protozoa), and it can also serve as a review for those already familiar with such methods.  In the first part of the exercise you will view a videotape, and in the second part you will perform many of the manipulations demonstrated on the tape, but without a live flame.  Therefore, this exercise is a simulation.  Following successful completion, arrangements can be made to repeat the steps with use of a real flame in the Cell Biology or Microbiology lab, under supervision of an instructor.


Exercise 1.  Obtain the videotape labeled “Aseptic technique” from the Tech Facility instructor and view it.  When finished, rewind it in the tape rewinder and return it to the instructor.


Exercise 2.   Obtain the box labeled “Aseptic technique” from the Tech Facility instructor, and examine its contents.  In it you will find:

(a) a bunsen burner (DO NOT TRY TO ATTACH IT TO THE GAS)

(b) an inoculation loop and needle

(c) a sterile pipette

(d) a capped erlenmeyer flask containing “medium”

(e) a tube rack with “sterile” capped tubes, some containing “cultures” and others containing “media” (see labels)

(f) a Petri dish containing an agar-based medium

(g) a bottle of “disinfectant”


Exercise 3.  Flaming the loop and inoculating a liquid culture

(a) Use a paper towel and some of the “disinfectant” to decontaminate your desktop work area.

(b) Plug the bunsen burner pump into an electrical outlet!  This will produce a simulated “flame”.

(c) Use the loop to remove a sample from one of the capped tubes labeled “culture”, following the video procedure.

(d) Transfer the loop sample into a “medium” tube (remember to “flame” the second tube, and flame the loop when finished)


 Exercise 4.  Flaming the loop and inoculating a semi-solid medium in a Petri dish

  (a) Plug the bunsen burner pump into an electrical outlet if not already plugged in!  This will produce a simulated “flame”.

 (b) Use the loop to remove a sample from one of the capped tubes labeled “culture”, following the video procedure.

 (c) Transfer the loop sample into a Petri dish, streaking the plate.  Remember to keep the Petri dish lid only slightly open, and above the dish bottom when working, as in the video. Flame the loop when finished.

  (d) When you feel confident about your technique, call over your peer supervisor and give a demonstration!


Exercise 5.  Transferring medium by pipette.

  (a) Obtain the erlenmeyer flask,a sterile pipette, and one of the capped tubes.

 (b) Use the pipette plus a pipette bulb (or other device) to transfer 2 ml of medium from the erlenmeyer flask to a culture tube.  Remember to “flame’ both the flask and the tube, as in the video.

 (c) Dispose of the pipette in the special waste container (red bag - biohazard) near the work area


FINALLY, CLEAN UP YOUR WORK AREA; PLEASE PUT ALL ITEMS BACK IN THE ORIGINAL BOX AND RETURN THE BOX TO ITS STORAGE PLACE.