BIOLOGY & SCIENCE: article : Streaking for Isolation of Bacterial Colonies on an Agar Medium

Thursday, March 12, 2009

article : Streaking for Isolation of Bacterial Colonies on an Agar Medium

Bacteria in natural circumstances are almost always found as mixtures of many species. For most purposes, it is necessary to isolate the various organisms in pure culture before they can be identified and studied. The most important technique for this purpose is "streaking out" on the surface of a solid nutrient medium, the principle being that a single organism, physically separated from others on the surface of the medium, will multiply and give rise to a localized colony of descendants. It is extremely important that you master this technique:

Sterilize a wire loop by heating it until red hot in a flame; allow it to cool for several seconds. Test for coolness by touching the agar at the edge of the plate.

Pick up a loopful of liquid inoculum or bacterial growth from the surface of an agar plate and, starting about one inch in from the edge of the plate, streak lightly back and forth with the loop flat, making close, parallel streaks back to the edge of the plate.

Sterilize the loop and cool again, then with the edge of the loop, lightly make another set of nearly parallel streaks about 1/8 inch apart, in one direction only, from the inoculated area to one side of the uninoculated area, so that about 1/2 the plate is now covered.

Flame and cool the loop again, and make another set of streaks in one direction, perpendicular to and crossing the second set of streaks, but avoiding the first set.

Note: A culture taken with a cotton swab (e.g., throat swab) can be rolled and rubbed back and forth across the plate. Streaking from this area is then continued with a wire loop, as above. Alternatively, material from the swab can be suspended in 1 ml of sterile broth, which is then cultured as above. To sample a dry surface (skin, dish, table, etc.), moisten a swab with sterile broth, and then use it to rub the surface. Solid material (soil, food, etc.) should be suspended in a small amount of sterile broth or peptone water, which is then streaked out; or, a dilution series may be made for an accurate count, as in food and water testing.