Philadelphia University

Faculty of Science
Department of Biotechnology & Genetic Engineering

General Microbiology Lab.5

Culture Transfer Methods

Culture Transfer Instruments

A pipette is an instrument often used to transfer aliquots of culture, to prepare serial dilutions of
microorganisms, and to dispense chemical reagents. Two types of measuring pipettes are
frequently used: the blow-out pipette (also called a serological pipette) and the to-deliver
pipette (Mohr measuring pipette). With the blow-out pipette, the final few drops of liquid must
be emptied in order to deliver the correct volume. With the to-deliver pipette, after the proper
amount of liquid has been delivered, liquid will remain in the tip of the pipette and should not be
eliminated. To fill a pipette, use a bulb or other mechanical device (figure 14.1c,d,e,f ). DO NOT
USE YOUR MOUTH. Draw the desired amount of fluid into the pipette. The volume is read at
the bottom of the meniscus. Often the mouth end of a pipette is carefully plugged with a small
piece of cotton before sterilization. This prevents cross-contamination of the bulb or mechanical
device of the pipette.
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Faculty of Science
Department of Biotechnology & Genetic Engineering

Inoculating needles and loops are used to aseptically

transfer microorganisms from broth, slant, or agar
cultures to other media. Both may consist of handles, a
shaft, and a turret, which holds a nickel chromium or
platinum wire. If the wire is straight, it is an inoculating
needle; if a loop is present, it is an inoculating loop.
Before using either, the end of the wire must be
sterilized by passing it slowly through the tip of the
flame from a Bunsen burner. When done correctly, all
parts of the wire will turn red with heat. The needle or
loop should then be used before it becomes
contaminated. After you have finished using an
inoculating loop or needle, it should be thoroughly
flame-sterilized.

Microorganisms are transferred from one culture medium to another by subculturing, using
specific procedures and aseptic technique. Asepsis means free from sepsis [a toxic condition
resulting from the presence of microorganisms. Since microorganisms are always present in the
laboratory, if aseptic technique is not followed, there is a good possibility that external
contamination will result and will interfere with the results. Proper aseptic technique also
protects the laboratory worker from contamination with the culture.

Principles for Isolation of Pure Cultures and Their Maintenance

Once discrete, well-separated colonies develop on the surface of the streak plate, selected ones
may be picked up with an inoculating needle and transferred to separate culture tubes, such as
tryptic soy agar slants (the type of agar will depend on the microorganism). Where possible,
bacteria from the center of a colony are transferred, because the center is less likely to be
contaminated than the edges. Each slant now represents the growth of a single species of
microorganism and is called a pure or stock culture. Short-term maintenance (generally
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Faculty of Science
Department of Biotechnology & Genetic Engineering

between one to three months) of aerobic bacteria can often be achieved by storing slant cultures
in the refrigerator at 4° to 10°C. The use of screw-cap tubes for these slants will minimize
desiccation during storage.

The best way to preserve many stock cultures for long periods is through lyophilization (freeze-
drying). This eliminates the need for periodic transfers and reduces the chance of mutations
occurring in the stock culture. In lyophilization, the bacterial culture is suspended in a sterile
solution of some protective medium such as milk, serum, or 3% lactose. Small amounts of the
thick suspension are transferred to vials and then quickly frozen in a dry-ice/alcohol mixture.
The frozen suspension is finally dried under vacuum while still frozen, and the vial sealed. These
sealed, desiccated cultures may often be stored for years. Strict anaerobes and some facultative
anaerobes will be injured by exposure to O2. They can often be maintained as agar stab cultures.
In this procedure, one allows a tube of the desired agar to solidify in an upright position and then
inoculates it by thrusting an inoculation needle coated with bacteria into the center of the agar.
 Philadelphia University
Faculty of Science
Department of Biotechnology & Genetic Engineering

The anaerobes will grow deep within the agar in the anaerobic environment it provides. After
suitable growth, the stab may be refrigerated.
Procedure for Culture Transfer Instruments and Techniques

Aseptic Technique:

1. Using a wax pencil, label the tube or plate to be inoculated with the date, your name, and the
name of the test microorganism (figure 14.3a).
2. Gently mix the primary culture tube in order to put the bacteria into a uniform suspension
(figure
14.3b).
3. Place the stock culture tube and the tube to be inoculated in the palm of one hand and secure
with the thumb. The tubes are then separated to form a V in the hand (figure 14.3c). They should
be held at an angle so that the open ends are not vertical and directly exposed to airborne
laboratory contaminants.
4. Using the other hand, flame the inoculating loop or needle over a Bunsen burner until the wire
becomes red-hot (figure 14.3d).
5. Using the same hand that is holding the inoculating loop, remove the caps from the two tubes,
hold them between your fingers, and briefly flame the necks of the tubes over a Bunsen burner
(figure 14.3e) by passing them through the flame. However, DO NOTALLOW THE TUBES TO
BECOME RED-HOT.
6. Cool the hot loop in the broth culture until it stops “hissing.” With the sterile inoculating loop,
transfer 1 drop of culture from the stock culture tube into the new broth tube. At this point, one
could also transfer to a glass slide, streak the surface of a slant, or streak the bacteria onto the
surface of a petri plate (figure 14.3f ). When picking up bacteria from a slant, cool the hot loop or
needle by holding it against the top of the slant until it stops “hissing.”
7. Reflame the neck of the tubes (figure 14.3g).
8. Recap the tubes (figure 14.3h).
9. Reflame or sterilize the loop or needle (figure 14.3i).
Procedure for Isolation of Pure Cultures and Their Maintenance

1. With a wax pencil, label the tryptic soy agar slants with the names of the respective bacteria.
Do the same for the broth tubes. Add your name and date.
2. Using aseptic technique, select a well-isolated colony for each of the three bacteria and pick
off as much of the center of the colony as possible with an inoculating loop. It may be necessary
to obtain material from more than one colony. Prepare a slant culture and a tryptic soy broth tube
for each of the bacteria. If screw-cap tubes are used, they must be loosened slightly before
incubation to keep the slant aerobic.
 Philadelphia University
Faculty of Science
Department of Biotechnology & Genetic Engineering

3. After incubating 24 to 48 hours, you should have three pure slant and three pure broth stock
cultures.
4. Observe the broth cultures.
5. Place the pure cultures in the refrigerator for later use.
1. With a wax pencil, label the tryptic soy agar slants with the names of the respective bacteria.
Do the same for the broth tubes. Add your name and date.
2. Using aseptic technique, select a well-isolated colony for each of the three bacteria and pick
off as much of the center of the colony as possible with an inoculating loop. It may be necessary
to obtain material from more than one colony. Prepare a slant culture and a tryptic soy broth tube
for each of the bacteria. If screw-cap tubes are used, they must be loosened slightly before
incubation to keep the slant aerobic
 Philadelphia University
Faculty of Science
Department of Biotechnology & Genetic Engineering

Plating Techniques
Spread-Plate Technique:
In natural habitats, bacteria usually grow together in populations containing a number of species.
In order to adequately study and characterize an individual bacterial species, one needs a pure
culture. The spread-plate technique is an easy, direct way of achieving this result. In this
technique, a small volume of dilute bacterial mixture containing 100 to 200 cells or less is
transferred to the center of an agar plate and is spread evenly over the surface with a sterile, L-
shaped glass rod. The glass rod is normally sterilized by dipping in alcohol and flamed to burn
off the alcohol. After incubation, some of the dispersed cells develop into isolated colonies. A
colony is a large number of bacterial cells on solid medium, which is visible to the naked eye as
a discrete entity. In this procedure, one assumes that a colony is derived from one cell and
therefore represents a clone of a pure culture. After a well-isolated colony has been identified, it
can then be picked up and streaked onto a fresh medium to obtain a pure culture.
Procedure:
1. Label the bottom of the agar medium plates with the name of the bacterium to be
inoculated, your name, and date.
2. Pipette 0.1 ml of the respective bacterial culture onto the center of the agar plate.
3. Dip the L-shaped glass rod into a beaker of ethanol and then tap the rod on the side of the
beaker to remove any excess ethanol.
4. Briefly pass the ethanol-soaked spreader through the flame to burn off the alcohol, and
allow it to cool inside the lid of a sterile petri plate.
5. Spread the bacterial sample evenly over the agar surface with the sterilized spreader,
making sure the entire surface of the plate has been covered. Also make sure you do not
touch the edge of the plate.
6. Invert the plates and incubate for 24 to 48 hours at room temperature or 30°C.

Note: An inoculated plate is always incubated in an inverted position to prevent

condensation from falling onto the surface of the plate and interfering with discrete colony
formation.
Streak- Plate Technique:

Isolated, pure colonies can also be obtained by the streak-plate technique. In this technique, the
bacterial mixture is transferred to the edge of an agar plate with an inoculating loop and then
 Philadelphia University
Faculty of Science
Department of Biotechnology & Genetic Engineering

streaked out over the surface in one of several patterns. At some point on the streaks, individual
cells will be removed from the loop as it glides along the agar surface and will give rise to
separate colonies. Again, one assumes that one colony comes from one cell. The key principle of
this method is that by streaking, a dilution gradient is established on the surface of the plate as
cells are deposited on the agar surface. Because of this gradient, confluent growth occurs on part
of the plate where the cells are not sufficiently separated, and individual, well isolated colonies
develop in other regions of the plate where few enough cells are deposited to form separate
colonies that can be seen with the naked eye. Cells from the new colony can then be picked up
with an inoculating needle and transferred to an agar slant or other suitable medium for
maintenance of the pure culture.
 Philadelphia University
Faculty of Science
Department of Biotechnology & Genetic Engineering

Pour-Plate Technique:

The pour-plate technique also will yield isolated colonies and has been extensively used with
bacteria and fungi. The original sample is diluted several times to reduce the microbial
population sufficiently to obtain separate colonies upon plating. The small volumes of several
diluted samples are added to sterile petri plates and mixed with liquid media agar that has been
cooled to about 48° to 50°C. Most bacteria and fungi will not be killed by the brief exposure to
the warm agar. After the agar has hardened, each cell is fixed in place and will form an
individual colony if the sample is dilute enough. Assuming no chaining or cell clusters, the total
number of colonies are equivalent to the number of viable microorganisms in the diluted sample.
To prepare pure cultures, colonies growing on the surface or subsurface can be inoculated into
fresh medium.

Pour-Plate Technique