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
1. 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.
2. 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 4: With one hand remove

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

8 :Incubate o/n at 37°C

4: Reflame metal 5,6,7: Rotate petri dish 90° Use 1st streak as
inoculating loop, inoculum for 2nd streak (only pass the loop
let cool through the 1st streak once). Repeat once
momentarily. 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.