Activity No.

3 ASEPTIC TECHNIQUES AND INOCULATION METHODS

Aseptic techniques involve the use of procedures that will prevent the contamination of the
specimen being worked on and the spread of microorganisms from the specimen to the
surroundings and to the handler. It is the practice of containing the microorganisms where
they rightfully belong.

Vessels containing sterile materials are kept closed except for the minimum time needed for
access. As one opens a vessel (e.g. removal of screw cap or cotton plug from test tube
containing specimen), the mouth must be passed briefly through the bunsen flame. Flaming is
also done immediately before closing the vessel. Take note that flaming must not be done if
the contents of the vessel are likely to catch fire.

The risk of contamination in the laboratory may be further reduced by treating the working
table top with a suitable disinfectant (e.g. 2.5 % hypochlorite) as well as filtering the air to
remove cells and spores of bacteria and fungi. Certain microbiological work require that it be
done in a biosafety cabinet (BSC) or clean bench. In a Class II laminar flow BSC, sterile (filtered)
air constantly flows onto the work surface. Work is conducted via an open panel in front of the
cabinet. Class II cabinets are common in college laboratories. A class III cabinet is a gas-tight
cabinet in which air is filtered before entry and before discharge to the environment. Work is
conducted via arm-length rubber gloves fitted into the front panel, and access to the interior of
the cabinet is via a separate two-door sterilization/disinfection chamber. Class III cabinets are
used when working with highly pathogenic microorganisms.

In most cases, microorganisms such as bacteria or fungi can be handled using an inoculating
loop or needle. A loop or needle is sterilized immediately before and after use by flaming. The
entire wire portion is heated to red hot in the non-luminous flame and is then allowed to cool.
Do not shake the instrument while cooling.

If a sterile loop is dipped into a suspension of microorganisms growing in a liquid medium and
withdrawn, the loop retains a small circular film of liquid containing a number of microbial cells,
such can be used as an inoculum. The size of this inoculum will depend on (a) concentration of
cells in the suspension and (b) the size of the wire loop which often carries 0.01 – 0.005 ml of
liquid. Smaller amount of liquid can be obtained using a needle as this picks up only a minute
volume of suspension, which adheres to the wire surface.

Loop or needle can also be used for picking up colonies growing on a solid medium. This is
done by bringing the loop or the tip of the needle into contact with the colony.

Further, spattering, which results in aerosol formation (NOTE: aerosol may contain viable
microorganisms) may occur when flaming the loop or needle with residual inoculum. Such may
be avoided by flaming the wire portion in the inner part of the flame.
Larger volumes of liquid may be handled by means of Pasteur pipettes or graduated pipettes.
Suction is obtained using an aspirator such as a rubber bulb. Mouth-pipetting MUST NEVER BE
USED. Pipettes are usually plugged with cotton at the region near the aspirator before
sterilization. This is to filter out microbial contaminants in the air coming from the aspirator
during use. Pipettes are usually sterilized inside metal canisters, and only the plugged end
should be held so as to avoid contaminating the rest of the pipette. Used pipettes are
immediately placed in a vessel containing disinfectant until they are safe to handle, when they
can be washed, sterilized and re-used.

METHODS OF INOCULATION

1. Inoculating a liquid medium

- inoculating loop or needle carrying the inoculum is dipped into the liquid medium,
moved slightly and then withdrawn.

2. Inoculating a solid plated medium

a. Clock streaking : this method is used when isolated colonies are required and the
inoculum is known to contain a large number of cells. In this method, the inoculum is
progressively 'thinned out' in such a way that individual, well separated cells are left
at some areas of the plate. Upon incubation, each well separated cell gives rise to an
individual colony.

b. Imprint method : this method may be employed if one wants to isolate

microorganisms on a surface of a material (e.g. hand surface or leaf surface). The
material is pressed over the surface of the solid medium for sometime. Upon
removal of the material, an imprint may be observed. On incubation, the distribution
of the microorganisms on the surface of the material may be traced.

c. Direct plate exposure : the agar plate is directly exposed to the surrounding air by
leaving the petri dish with the nutrient medium open for a certain period of time.
This method may be employed if one wants to sample microorganisms from aerosol
or from the surrounding air.

d. Spread plate : a small volume of liquid inoculum is spread over the surface of a solid
medium using a sterile bent glass rod, also called a hockey stick.

e. Flood plate : this is done by flooding the surface of a solid medium with a liquid
inoculum and withdrawing excess inoculum with a sterile Pasteur pipette.

After proper incubation, both the spread plate and flood plate will give rise to a lawn plate, a
plate in which the surface of the medium is covered with a layer of confluent growth.
 f. Pour plate method : In this method, an inoculum is delivered into empty, sterile plate
using a sterile graduated pipette (if a measured amount of inoculum is needed) or
streaked out with a loop. Sterile, melted nutrient agar medium is then poured until
the bottom of the plate is completely covered. The plate is swirled gently to evenly
distribute the inoculum into the agar. The agar plate is then left to solidify and then
incubated. In this method, the growth of microorganisms will not be confined on the
surface of the agar plate but also on the subsurface.

A plate may sometimes be inoculated with sterile cotton swabs. A sterile swab is used for
example for sampling microorganisms at a given site (e.g. throat). After exposure, the swab is
drawn lightly on the surface of the agar. Swab inoculation may be done either by inoculating
the entire surface of the agar plate to produce a lawn plate, or by inoculating a small peripheral
area, and further distributing the inoculum by streaking using an inoculating loop or needle.

If the site to be sampled is a dry surface, the tip of the cotton swab is moistened with sterile
normal saline solution (NSS) or sterile broth medium.

3. Inoculating a solid tube medium

a. Streaking : this method is used to inoculate the surface of slants. One may employ an
inoculating loop, needle or swab for this purpose.

b. Stabbing : this method is used when one has to inoculate the deeper, microaerobic or
anaerobic portion of a butt. A needle is preferred for this inoculation method. The
needle containing the inoculum is plunged vertically unto the butt.

Reference :

Singleton, P. 1999. Bacteria in Biology, Biotechnology and Medicine.

Wiley and Sons, pp.365-370.