Transcript lab3 MIC 140

ASEPTIC TECHNIQUE
• Removing inoculum from a broth
culture
(organisms growing in a liquid medium)
• Hold the culture tube in one hand and in
your other hand hold the sterilized
inoculating loop
• Remove the cap of the pure culture tube
with the little finger of your loop hand
• 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
• Again flame the lip of the culture tube
and Replace the cap
• Transferring the inoculum into a broth tube
Pick up the sterile broth tube and remove the cap with the little finger
• flame the lip of the broth tube
Place the loopful of inoculum into the broth
and withdraw the loop
• Again flame the lip of the tube
Replace the cap
• 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
• Scrape off a small amount of the
organisms and close the lid
• Transferring the inoculum into a broth tube
Pick up the sterile broth tube and remove the cap with the little finger
• flame the lip of the broth tube
Place the loopful of inoculum into the broth
and withdraw the loop
• Again flame the lip of the tube
Replace the cap
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 2448 hours.
6. Observe the slant for growth.
Inoculated Agar Slant, after
incubation
• 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
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
.
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!
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
• 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
• 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.
• Place the plate in a 37o C incubator for
24-48 hours.
Quaetrant streak
1
Nice isolation!
2
4
1
2
3
Streak pattern
4
3
Bacterial growth pattern
Starting with a mixed
culture, isolation of
different species may
be obtained by streaking
Contamination of a streak plate results from leaving the
plate open too long or not shielding properly with the lid.
Correct procedure
Which streak plate culture started as a pure culture. How can you tell?
Answer: the one on the right, because all colonies look alike.