1. ASEPTIC TECHNIQUE Removing inoculum from a broth culture
(organisms growing in a liquid medium)

2. Hold the culture tube in one hand and in your other hand hold the sterilized inoculating loop

3. Remove the cap of the pure culture tube with the little finger of your loop hand

4. Keeping the culture tube at an angle, insert the inoculating loop and remove a loopful of inoculum
Remove a loopfull of bacteria from your pure culture

5. Again flame the lip of the culture tube
and Replace the cap

6. flame the lip of the culture tube

7. Transferring the inoculum into a broth tube
Pick up the sterile broth tube and remove the cap with the little finger

8. flame the lip of the broth tube

9. Place the loopful of inoculum into the broth and withdraw the loop

10. Again flame the lip of the tube
Replace the cap

11. Removing inoculum from a plate
organisms growing on an agar surface in a petri plate
Sterilize the inoculating loop in the flame
Lift the lid of the culture plate and stab the loop into
the agar away from any growth to cool the loop

12. Scrape off a small amount
of the organisms and close
the lid

14. Inoculating an Agar Slant
1.Label the sterile nutrient agar slant with the source of the culture and your
initials.
2. Sterilize the loop.
3. Using appropriate aseptic technique, remove a loopful of broth from the culture tube.
4. Insert the loop into the sterile agar slant tube and starting at the base of
the slant, draw the loop up the slant. Do not penetrate the agar. Sterilize
the loop.
5. Incubate the slant at 37o C for hours.
6. Observe the slant for growth.

17. Inoculated Agar Slant, after incubation

18. microorganisms exist in nature as mixed populations(A mixed culture contains two or more bacterial species )However, to study microorganisms in the lab we must have them in the form of a pure culture

19. assumed to be a pure culture
Streak plates allow for the growth of isolated colonies on the surface of the
agar. An isolated colony is a colony that is not touching any other colonies and is
assumed to be a pure culture .

20. You’re never too old to streak!
STREAK PLATE METHOD OF ISOLATION
Purpose of streaking:  To obtain pure, isolated colonies.
Principle:  By spreading a large amount of bacteria over the large surface area of a plate, the amount of bacteria is diluted until individual cells are spread on the surface of the plate.  Each individual cell grows into a single colony.
You’re never too old to streak!

21. Streak Plate
Label the sterile nutrient agar plate with the source of the culture and your initials.
. Sterilize the loop.
. Using appropriate aseptic technique, remove a loopful from the mixed culture plate.
. Lift the agar plate from the lid and streak about half of the plate. The loop should be parallel to the agar surface
to prevent digging into the agar

22. Return the plate to the lid. Sterilize the loop
Return the plate to the lid. Sterilize the loop. Lift the agar plate and make
one streak into the inoculated portion of the plate. Finish by streaking
about one-fourth of the uninoculated plate

23. Return the plate to the lid. Sterilize the loop
Return the plate to the lid. Sterilize the loop. Lift the agar plate and make
one streak into the second inoculated portion of the plate. Finish by
streaking the remaining one-fourth of the uninoculated plate
Sterilize the loop.

24. Place the plate in a 37o C incubator for 24-48 hours.

25. Quaetrant streak

26. Streak pattern 1 2 3 4
Bacterial growth pattern
Nice isolation!

27. Starting with a mixed culture, isolation of different species may be obtained by streaking

28. Contamination of a streak plate results from leaving the plate open too long or not shielding properly with the lid.
Correct procedure

29. Which streak plate culture started as a pure culture. How can you tell?
Answer: the one on the right, because all colonies look alike.