Microbiology Laboratory

Exercise 3: Aseptic Transfer of Cultures

Pure cultures are an indispensable aid in the study of microorganisms. Hence, all
attempts must be made to prevent inclusion of unwanted microorganisms
(contaminants). This is made possible by practicing aseptic techniques in the
laboratory. As long as asepsis is observed in every transfer, no problems should
arise in the maintenance and study of pure cultures.

OBJECTIVES:
To acquire and develop skills in the aseptic transfer of culture of
microorganisms to the different form of prepared media.

METHODS:
IMPORTANT: Disinfect the surface of your working area before starting the
experiment!

A. Flame-sterilization of the wire loop / Needle

1.

Hold the loop / needle almost vertically over the flame and heat both the
wire and the lower part of the handle. Make sure the entire wire gets redhot.

2. Allow the loop / needle to cool for a few seconds before touching the
inoculums. Do not sway the loop / needle nor let it come in contact with
anything.
3.

After transfer of culture has been completed, re sterilize the loop / needle
as in n

4. Re peat steps 1 to 3 every time transfers of new cultures are made. DO

NOT forget to cool the loop / needle before allowing it to come in contact
with the culture or the medium

B.Transferring Culture to Tube Media.

1. Flame- sterilize the loop / needle as in A. 1 and A. 2.

2. Hold the tube containing the culture with the left hand and remove the
plug from the test tube with the little finger of the right hand. DO NOT PUT
THE PLUG DOWN AND WORK AS CLOSELY AS POSSIBLE NEAR THE FLAME
THROUGHOUT THE PROCESS OF TRANSFERRING.
3. Flame the mouth of the tube passing it back and forth through the flame
(about 4-5 times).
4. Remove the loopful of the inoculums (if in a broth) or small amount of
growth (if in a solid medium).
5. Reflame the mouth of the tube, return the plug and put the culture back in
the rack. Get the tube of fresh medium.
6. Transfer the cultures into the ff. tubes of uninoculated media:
Slant moves the loop down to the end and draw the loop upward in a
zigzag option or make a single linear strike. Transfer of mold culture is
made by point inoculation (stabling the medium with the needle once to
deposit the inoculums) See Fig 3.4

a. Deep / Stab stab the loop deep into the medium.

b. Broth immerse the loop into the broth and gently shake to deposit
the cells.
7. After transfer is accomplished, add the following informations into the
label of the inoculated tubes:
a. Organism inoculated
b. Date of inoculation

C.Transferring Culture to Plated Media

1. Divide the bottom of the petri dish into 4 sectors and mark as in:

2. When you have mastered the Streaking technique, you need not to do
this anymore. With the loop (for bacteria, yeasts) or needle (for molds)
containing the inoculums on your right hand, hold the petri dish with your
left hand and lift its cover. See Fig. 3.5

3. Introduce the inoculums on first quandrant and follow the streaking

pattern Mold creatures are also transferred to plated media by point
inoculation in 2 3 areas.
4.