Sterile technique When handling E. coli and other bacteria it is essential that the live cultures do not become contaminated with other bacteria or fungi. The set of procedures used to accomplish this are known as “sterile technique” General Points 1: Keep vials or plates containing bacteria open for a minimum amount of time. 2: Use sterilized instruments when handling the bacteria 3: Discard all bacteria in “biohazardous” waste - this will be destroyed later 4: When using an open flame never leave it unattended

Streak Plate method to Purify Single bacteria Principle This is essentially a method to dilute the number of organisms, decreasing the density - individual colonies to be isolated from other colonies. Each colony is "pure," since theoretically, the colony began with an individual cell Begin with inoculating the first, or primary, quadrant of the agar plate. Use a light touch. Don't penetrate or scrape the agar surface. Cover plate with lid. Flame the loop, cool by touching an uninoculated portion of the surface. 3. Now rotate the plate. Open lid and streak again, remember: you are picking up growth from quadrant one, and using this as your inoculum for quadrant two. 4. Flame loop; rotate plate, and repeat procedure for quadrants three and four.

Performing a Plate Streak I 2-3: Using sterile technique transfer a loop of bacterial culture or single colony onto loop 4: With one hand remove lid of dish. With other hand lightly brush the loop back and forth on one quadrant of the dish 1: Flame metal inoculating loop, let cool momentarily.

Performing a Plate Streak II 8 :Incubate o/n at 37°C 4: Reflame metal inoculating loop, let cool momentarily. 5,6,7: Rotate petri dish 90° Use 1st streak as inoculum for 2nd streak (only pass the loop through the 1st streak once). Repeat once more rotating dish 90° and sterilizing loop again

Plate Streak Method This is an example of a good streak for isolation using the "four corners" method.

This is not a great streak plate but it is serviceable, as there are a few isolated colonies. - would have been better if the loop had been flamed between each sector. This is an example of how NOT to streak for isolation. Scribbling is not streaking, and most likely will not result in isolated colonies.