In working with living cultures of organisms, it is essential to observe a
procedure known as aseptic (sterile) technique. The goals of this
procedure are twofold:
to prevent contamination of the culture
prevent spread of culture organisms to the lab personnel or the environment.
The tubes of growing bacteria provided to you are stock cultures of
common organisms. These cultures should each be pure cultures (only
one species of organism in the culture). Our goal today is to allow you to
start your own cultures from these stock cultures, maintaining the purity
(avoiding contamination with other species). The organisms you will work with
Escherichia coli (E. coli) is a normal resident in the bowel
of man and animals. It is a short rod. Easily grown under
laboratory conditions and has been studied extensively. Research with E.
coli has led to advances in our understanding of cellular metabolism as
well as genetics. Genetic engineering using this organism has led to the
bulk production of heretofore to obtain human cell products such as insulin,
somototropin, and interferon.
Bacillus subtilis (B. sub.) is a common soil organism.
Since it is an endospore-forming long rod, it survives many
Staphylococcus epidermidis (S. epi.) colonizes the skin surface.
It tolerates the high salt concentration and relative dryness of such an
In order to appreciate the aseptic technique, you must consider any organism,
no matter how common, to be a potential pathogen (agent of disease).
Indeed, in great enough numbers and achieving the appropriate portal of entry,
almost any bacterium can induce an infection.
The organisms grown in pure culture for laboratory study are grown on a growth
medium, which may be a liquid broth or a solid agar slant or plate.
A particular medium may be designed to contain only the minimum requirements for
growth of a specific organism. This would be called “ minimal media”
for that organism. If one needs to encourage rapid growth of a very small
sample of organisms, an enriched medium may be used, reinforced with growth
factors required by fastidious organisms. Typically, common
organisms are grown on a standardized beef extract or protein base containing
requirements for most bacterial species. Nutrient media and tryptic
soy media are examples.
READ THIS ENTIRE PROCEDURE AND WATCH IT DEMONSTRATED. PRACTICE WITH
EMPTY TUBES BEFORE ATTEMPTING THE TRANSFER!!!
You and your partner will be given three test tubes of sterile medium and
three plates of agar.
- First, label the tubes with the following information: Name, date,
and the name of organism to be inoculated.
- Place the tubes in a rack and turn on the Bacti-Cinerator nearest
your lab team. It should heat for ten minutes before use. Get
the inoculation loop from your drawer. (Make sure the loop
has an insulated handle)
- The inoculation loop is to be inserted into the chamber without touching
the sides. It should be deep enough to heat the loop end of the
holder. ( This end of the holder is often a source of
contamination). Hold the loop in the chamber long enough for the
wire to turn red along it’s entire length. This should take about
ten seconds. As the wire glows red, it is sterilized. The
procedure just described applies to any part of this lab manual wherein
the phrase “heat-sterilize” the loop occurs. Do not turn
the inoculation loop handle loose while the loop is inside the chamber.
Such a “bad-habit” of resting the loop within the chamber causes the
handle to heat to burning temperatures and also damages the chamber.
The Bacti-Cinerator may be left on for the duration of the lab as long as
heat- sterilization is required.
- Choose a stock culture and a correspondingly labeled sterile tube.
Hold both in the left hand, with the culture closest to you.
(Reverse these instructions if you are left-handed). The instructor
- Loosen the caps until they will just lift off. Do not uncover the
tubes at this point.
- Hold the inoculation loop in your right hand, heat sterilize the wire.
Angle the wire so that it glows red through its entire length.
- The loop is now sterile. Let it cool a few seconds.
- Crook the little finger of the right hand around the cap of the stock culture
and the third or fourth finger around the cap of the sterile tube.
- Lift both simultaneously from the tubes, angle the tops of the tubes
into the mouth of the Bacti-Cinerator. This procedure hits the air
at the neck of the tubes so that environmental organisms do not fall into
and contaminate the tubes. Keep the caps in place between your
fingers, taking care not to palm them or turn them up. (Never put
the caps down on the lab table, this would contaminate them and also the
- Lower the now-cooled loop just below the surface of a broth stock
culture, or gently touch the loop to a bacterial colony if working from a
stock agar slant. You are taking a tiny sample of organisms from the stock
culture. This sample is referred to as the inoculum.
- Withdraw the loop without touching the sides of the tube.
- Move the loop directly into the adjacent sterile tube.
- Lower the loop below the surface, shake gently, and withdraw as before.
- Reheat the tube tops, then recap.
- Heat-sterilize the loop, let it cool enough to put down without burning
- Tighten the caps fully, then loosen by one turn and return the tubes to
General inoculation of a Petri dish containing agar.
- Label the plate with the same information used to label your tubes.
Write this information directly on the bottom of the plate, around the edge
using a wax pencil or marker.
- Heat sterilize your loop and remove the cap from the stock culture.
- Heat the top of the tube and reheat your loop is necessary.
- Enter the tube, allowing enough time for the loop to cool, enter the
broth and remove your inoculum.
- Heat the top of the tube again, recap the tube, and place it to the side
in a test tube rack.
- Open the Petri dish by removing the top. This should only be done when
you are ready to inoculate the plate. Air can carry contaminates to the
surface of the agar if the plate is left open.
- Gentle place the loop on the surface of the agar and move it from side
to side while also moving down the length of the plate.
- When finished, replace the lid of the dish, sterilize your loop, and
place the plates in the appropriate rack INVERTED to allow for proper
Some errors commonly made involve handling of the
loop. If it has not cooled enough before touching broth or agar, a
sizzling sound will be heard. This produces aerosols of living
bacteria, which contaminate the environment, and can reduce the chance of
viability of the transferred specimen. If the loop drags against the glass
on withdrawal, a strong vibration sprays living bacteria into the air. Touching
the agar with a loop that is too hot will cause burn scars to appear on the
surface and make streaking the plate difficult. Applying too much pressure to
the agar surface can lead to digging or pitting of the agar.
When you finish, place your cultures in the common
rack (designated by the instructor) for incubation at 37 degrees C.
The cultures will be incubated for 48 hours and you will use them for the next
two lab sessions in order to learn staining techniques.
FOR TESTING PURPOSES: In order to test the quality of aseptic
technique, a sterile transfer may be made between two sterile media
tubes. If no growth occurs in either tube following 48 hours of
incubation, the technique is adequate.