This document provides instructions for several microbiology techniques, including how to hold an inoculating loop, streak plating bacteria to isolate colonies, transferring bacteria between culture tubes or broths, and measuring zones of inhibition. Streak plating involves spreading bacteria in a sparse pattern on an agar plate to isolate individual colonies after incubation. Transferring involves flaming loops or needles used to move bacteria between tubes. Measuring zones of inhibition analyzes the effect of antibiotics on bacterial growth.

1. Microbiology
Techniques
August 17, 2013

2. Media Names
 Nutrient
Agar – NA
 Potato Dextrose Agar - PDA

3. How to hold an Inoculating Loop

4. Flaming the Loop

5. Streak Plate

6. Streak Plate
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Streak plating is used to
isolate a single type of
bacteria.
•
This technique spreads out
original “parent bacteria”
in a sparse pattern that
,after growth, results in
individual colonies.
•
After incubation, the 4th
quadrant of your plate
should have dots.
•
These small “dots” are
individual colonies, and
represent millions of
bacteria of the same type.
* IMPORTANT!!!: Be very gentle when
streaking the sample onto the plate. Try not
to make a hole on the surface of the
medium with your inoculation loop.

7. Transfer to tubes

8. Flaming tubes

9. Streaking a slant

10. Transfer
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Steps for Transfer of Broth to Broth
Hold loop or needle with dominant hand( right )
Flame the loop
Hold culture tube in left hand
Remove red cap with pinkie of right hand
Flame mouth of culture tube
Place loop into broth( water)
Flame mouth of culture tube and close
Open culture tube with broth( should be labeled)
Dip loop into new broth and mix
Flame mouth of tube and close
Flame loop
Place to the side of your rack

11. ASEPTIC PROCECURE FOR
MICROBE REMOVAL
http://www.youtube.com/watch?v=0odxJy0nR9s&feature=related

13. Streaking and flaming
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Flame the loop to sterilize it and let cool.
Position the plate so that the spot of inoculum is nearest the hand
not holding the loop (the opposite hand).
Lift the plate lid with the opposite hand; just enough to get the loop
inside and touch the loop to the inoculum spot. It is often helpful to
treat the inoculating loop as if it were a pencil - steadying the loop
by resting the heel of the hand against the lab bench.
Move the loop back and forth across the spot and then gradually
continue toward the center of the plate as you sweep back and
forth. Use a very gentle and even pressure.
When creating each phase, do not worry about keeping each pass
across the plate separate from previous ones.
When about 30% of the plate has been covered by the first
streaking phase, remove the loop and flame sterilize it.
Repeat the above procedure for the second phase, but this time pick
up some inoculum by crossing into the first phase 2-3 times and then
not passing into it again.
Repeat as necessary for the third and fourth phases. After
streaking the plate, flame sterilize the loop before setting it down.

14. Measuring Zone of Inhibition

16. END