1993 Woodrow Wilson Biology Institute
In some cases one must spread a number of cells on a plate to grow into colonies. The cells must be spread out so that the colonies will be separate and easily counted after incubation and growth. A sterile glass spreader (also known as a rake or a hockey stick) is used to smooth the cells evenly across the top of the agar.
Spreaders are easily made by heating and bending a 20 centimeter length of 3 mm diameter glass rod. Using a Bunsen burner, both ends are flame-polished, then the rod is heated and bent approximately three centimeters from one end to form an angle.
All of the following steps must be done using proper sterile technique to avoid contamination by unknown and unwanted microorganisms. Using a micropipette, a known volume of cells is removed from a test tube containing the microbial culture (either original culture or dilutions of the original). The small volume of cells is deposited onto the surface of the agar in a Petri dish. The spreader is sterilized by briefly dipping it in alcohol and passing it through the flame of a Bunsen burner. (Note: the Bunsen burner is not used to heat sterilize the rake; it is used only for igniting the ethanol.) After the spreader has been flamed, it should be moved through the air as little as possible. The cover of the Petri dish is lifted just enough to get the spreader under it and into the dish and the spreader is placed momentarily on the agar (NOT on the pool of microbial solution) to cool the glass. (Touching the hot spreader to the microbial solution may kill cells.) Next, the spreader is gently pushed two or three times clockwise around the dish, and then several times counterclockwise. Pressing too hard will cause the microorganisms to collect at the edge of the spreader, resulting in uneven distribution.