E XE R CI S E

8
MICROBIOLOGY

Aseptic Technique

Culture Tube Flaming and Inoculation Prior to

Learning Outcomes inserting a cooled loop or needle into a culture tube,
the cap is removed and the mouth of the tube may be
After completing this exercise, you should be able to
flamed. If the tube is a broth tube, the loop is inserted
1. Aseptically transfer a bacterial culture from one into the tube and twisted several times to ensure that
broth tube to a new broth tube. the organisms on the loop are delivered to the liquid.
2. Aseptically transfer a bacterial culture from an agar If the tube is an agar slant, the surface of the slant is
slant to a new agar slant. inoculated by drawing the loop up the surface of the
3. Aseptically transfer a bacterial colony from an agar slant from the bottom of the slant to its top. For stab
plate to an agar slant. cultures, a needle is inserted into the agar medium by
stabbing it into the agar. After the culture is inocu-
lated, the mouth of the tube may be reflamed and the
tube is recapped.
The use of aseptic technique insures that no contami-
nating organisms are introduced into culture materi- Petri Plate Inoculations Loops are used to inocu-
als when the latter are inoculated or handled in some late or streak petri plates. The plate cover is raised and
manner. It also insures that organisms that are being held diagonally over the plate to protect the surface
handled do not contaminate the handler or others who from any contamination in the air. The loop contain-
may be present. And its use means that no contamina- ing the inoculum is then streaked gently over the sur-
tion remains after you have worked with cultures. face of the agar. It is important not to gouge or disturb
As you work with these procedures, with time, the surface of the agar with the loop. The cover is
they will become routine and second nature to you. replaced and the loop is flamed.
You will automatically know that a set of procedures
outlined below will be used when dealing with cultures Final Flaming of the Loop or Needle After the
of microorganisms. This may involve the transfer of inoculation is complete, the loop or needle is flamed
a broth culture to a plate for streaking, or inoculating to destroy any organisms that remain on these imple-
an isolated colony from a plate onto a slant culture to ments. The loop or needle is then returned to its
prepare a stock culture. It may also involve inoculating receptacle for storage. It should never be placed on
many tubes of media and agar plates from a stock cul- the desk surface.
ture in order to characterize and identify an unknown
bacterium. Ensuring that only the desired organism is Final Disinfection of the Work Area When all work
transferred in each inoculation is of paramount impor- for the day is complete, the work area is treated with dis-
tance in the identification process. The general proce- infectant to insure that any organism that might have been
dure for aseptic technique follows. deposited during any of the procedures is killed.
To gain some practice in aseptic transfer of bacte-
Work Area Disinfection The work area is first treated rial cultures, three simple transfers will be performed
with a disinfectant to kill any microorganisms that may in this exercise:
be present. This process destroys vegetative cells and 1. broth culture to broth tube
viruses but may not destroy endospores. 2. agar slant culture to an agar slant and
Loops and Needles The transfer of cultures will be 3. agar plate to an agar slant.
achieved using inoculating loops and needles. These
implements must be sterilized before transferring any Transfer from Broth Culture to
culture. A loop or needle is sterilized by inserting it Another Broth
into a Bunsen burner or incinerator flame until it is
red-hot. This will incinerate any contaminating organ- Do a broth tube to broth tube inoculation using the fol-
isms that may be present. Allow the loop to cool com- lowing technique. Figure 8.1 illustrates the procedure
pletely before picking up inoculum. This will ensure for removing organisms from a culture, and figure 8.2
that viable cells are transferred. shows how to inoculate a tube of sterile broth.

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EXERCISE 8 Aseptic Technique

2. With a marking pen, label a tube of sterile nutrient

Materials broth with your initials and E. coli.
• broth culture of Escherichia coli 3. Sterilize your inoculating loop by flaming it until
• tubes of sterile nutrient broth it becomes bright red. The entire wire must be
• inoculating loop heated. See illustration 1, figure 8.1.
• Bunsen burner or incinerator 4. Using your free hand, gently shake the tube to
• disinfectant for desktop and paper towels disperse the culture (illustration 2, figure 8.1).
• marking pen 5. Grasp the tube cap with the little finger of your
hand holding the inoculating loop and remove
1. Prepare your desktop by swabbing down its surface it from the tube. Flame the mouth of the tube,
with a disinfectant. Use a sponge or paper towels.

(1) Inoculating loop is heated until it is (2) Organisms in culture are dispersed by (3) Tube enclosure is removed and mouth
red-hot. shaking tube. of tube is amed.

(4) A loopful of organisms is removed (5) Loop is removed from culture and tube (6) Tube enclosure is returned to tube.
from tube. mouth is amed.

Figure 8.1 Procedure for removing organisms from a broth culture with inoculating loop.

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 Aseptic Technique EXERCISE 8

as shown in illustration 3, figure 8.1. Note: if an 9. Without flaming the loop, insert it into the sterile
incinerator is used, the tube is not flamed. broth, inoculating it (illustration 2, figure 8.2). To
6. Insert the inoculating loop into the culture (illus- disperse the organisms into the medium, move the
tration 4, figure 8.1). loop back and forth in the tube.
7. Remove the loop containing the culture, flame 10. Remove the loop from the tube and flame the
the mouth of the tube again (illustration 5, mouth (illustration 3, figure 8.2). Replace the cap
figure 8.1), and recap the tube (illustration 6). on the tube (illustration 4, figure 8.2).
Place the culture tube back on the test-tube rack. 11. Sterilize the loop by flaming it (illustration 5,
8. Grasp a tube of sterile nutrient broth with figure 8.2). Return the loop to its container.
your free hand, carefully remove the cap with 12. Incubate the culture you just inoculated at 37 C
your little finger, and flame the mouth of this tube for 24– 48 hours.
(illustration 1, figure 8.2).

(1) Cap is removed from sterile broth and (2) Sterile, cooled loop is inserted into tube (3) Loop is removed from broth and
tube mouth is amed. of sterile broth. tube mouth is amed.

(4) Tube enclosure is returned to tube. (5) Loop is amed and returned to
receptacle.

Figure 8.2 Procedure for inoculating a nutrient broth.

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EXERCISE 8 Aseptic Technique

the loop (illustration 3, figure 8.3) and remove the

Transfer of Bacteria from a Slant
loop from the tube.
To inoculate a sterile nutrient agar slant from an agar 6. Flame the mouth of the tube (illustrations 4 and 5,
slant culture, use the following procedure. Figure 8.3 figure 8.3) and replace the cap, being careful not
illustrates the entire process. to burn your hand. Return tube to rack.
7. Pick up a sterile nutrient agar slant with your free
hand, remove the cap with your little finger as
Materials before, and flame the mouth of the tube (illustra-
• agar slant culture of E. coli tion 6, figure 8.3).
• sterile nutrient agar slant 8. Without flaming the loop containing the culture,
• inoculating loop insert the loop into the tube and gently inoculate
• Bunsen burner or incinerator the surface of the slant by moving the loop back
• marking pen and forth over the agar surface, while moving up
the surface of the slant (illustration 7, figure 8.3).
1. If you have not already done so, prepare your This should involve a type of serpentine or zig-
desktop by swabbing down its surface with a zag motion.
disinfectant. 9. Remove the loop, flame the mouth of the tube,
2. With a marking pen, label a tube of nutrient agar and recap the tube (illustration 8, figure 8.3).
slant with your initials and E. coli. Replace the tube in the rack.
3. Sterilize your inoculating loop by holding it 10. Flame the loop, heating the entire wire to red-hot
over the flame of a Bunsen burner until it becomes (illustration 9, figure 8.3), allow to cool, and place
bright red (illustration 1, figure 8.3). The entire the loop in its container.
wire must be heated. Allow the loop to cool 11. Incubate the inoculated agar slant at 37 C for
completely. 24–48 hours.
4. Using your free hand, pick up the slant culture of
E. coli and remove the cap using the little finger Working with Agar Plates
of the hand that is holding the loop (illustration 2,
figure 8.3). (Inoculating a slant from a petri plate)
5. Flame the mouth of the tube and insert the cooled The transfer of organisms from colonies on agar
loop into the tube. Pick up some of the culture on plates to slants or broth tubes is very similar to the

(1) Inoculating loop is heated until it is (2) Cap is removed from slant culture (3) Organism is picked up from slant
red-hot. and tube mouth is heated. with inoculating loop.

continued
Figure 8.3 Procedure for inoculating a nutrient agar slant from a slant culture.

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 Aseptic Technique EXERCISE 8

(4) Mouth of tube is amed. Inoculating (5) Slant culture is recapped and returned (6) Tube of sterile agar slant is
loop is not amed. to test-tube rack. uncapped and mouth is amed.

(7) Slant surface is streaked with (8) Tube mouth is amed, recapped, and (9) Loop is amed red-hot and returned
un amed loop in a zigzag motion. incubated. to receptacle.

Figure 8.3 (continued)

procedures used in the last two transfers (broth to cover should be only partially opened as shown in illus-
broth and slant to slant). The following rules should tration 2, figure 8.4.
be observed:
Flaming Procedures Inoculating loops or needles
Loops and Needles Loops are routinely used when must be flamed in the same manner that you used
streaking agar plates and slants. When used properly, a when working with previous tubes. One difference when
loop will not gouge or tear the agar surface. Needles are working with plates is that plates are never flamed!
used in transfers involving stab cultures.
Plate Labeling and Incubation Petri plates containing
Plate Handling Media in plates must always be pro- inoculated media are labeled on the bottom of the plate.
tected against contamination. To prevent exposure to Inoculated plates are almost always incubated upside
air contamination, covers should always be left closed. down. This prevents moisture from condensing on the
When organisms are removed from a plate culture, the agar surface and spreading the inoculated organisms.

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EXERCISE 8 Aseptic Technique

(1) Inoculating loop is heated until it is (2) With free hand, raise the lid of the petri plate just enough
red-hot. to access a colony to pick up a loopful of organisms.

(3) After aming the mouth of a sterile (4) Flame the mouth of the tube and (5) Flame the inoculating loop and
slant, streak its surface. recap the tube. return it to receptacle.

Figure 8.4 Procedure for inoculating a nutrient agar slant from an agar plate.

To transfer organisms from a petri plate to an agar 4. As shown in illustration 2, figure 8.4, raise the lid
slant, use the following procedure: of a petri plate sufficiently to access a colony with
your sterile loop.
Materials Do not gouge the agar with your loop as you
pick up organisms. Simply allow the loop to gently
• nutrient agar plate with bacterial colonies
glide over the gelatin-like surface of the agar. Do
• sterile nutrient agar slant
not completely remove the lid while inoculating or
• inoculating loop
removing organisms from the agar plate. This will
• marking pen
expose the agar surface to air and potential contami-
1. If you have not done so, swab your work area with nation. Always close the lid once you have removed
disinfectant. Allow area to dry. organisms from the plate.
2. Label a sterile nutrient agar slant with your name 5. With your free hand, pick up the sterile nutrient
and organism to be transferred. agar slant tube. Remove the cap by grasping the
3. Flame an inoculating loop until it is red-hot (illus- cap with the little finger of the hand that is hold-
tration 1, figure 8.4). Allow the loop to cool. ing the loop.

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 Aseptic Technique EXERCISE 8

6. Flame the mouth of the tube and insert the loop 8. Flame the loop (illustration 5, figure 8.4) and
into the tube to inoculate the surface of the place it in its container.
slant, using a serpentine motion (illustration 3, 9. Incubate the nutrient agar slant at 37 C for 24–
figure 8.4). Avoid disrupting the agar surface 48 hours.
with the loop.
7. Remove the loop from the tube and flame the Results
mouth of the tube. Replace the cap on the tube
(illustration 4, figure 8.4). Examine all three tubes and record your results in
Laboratory Report 8.

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