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All agar plates are incubated UPSIDE DOWN (exceptions will be pointed out occasionally).
WHY?
1. It reduces bacterial contamination since the bacteria, even if they get into the plate in
between the lid and bottom, would have to go UP to get to the agar.
2. It reduces the possibility of water condensation that may be on the lid dropping onto the
agar, causing fluid to run across the agar medium.
By now you know what media is: it is the nutrient-rich material that provides food to the
microbes. There are 3 forms of media:
In addition to nutrients and growth factors, and perhaps agar, there are additives such as
NaCl salt, pH buffers, and pH indicators that allow biochemical reactions to be identified.
In this exercise you will learn how to subculture bacteria, using a variety of culture media as
your inocula sources and as your new culture media. Different species of bacteria will be
used so that you can become familiar with different growth patterns. You will also have a
mixed culture with 2 species in order to learn how to best separate and isolate bacterial
species.
THE OBJECTIVES:
Subculture bacteria in/on sterile media of various forms.
Eliminate potential contamination of bacterial cultures by using aseptic technique.
Practice hand coordination required in good transfer techniques.
Identify different ways by which bacteria grow in culturein agar deeps, on agar slants, on
agar plates, in broths.
Streak out cultures for isolation and identify colonies.
MATERIALS NEEDED:
set of cultures for the table:
a TSA (trypticase soy agar) slant culture of Bacillus subtilis
a TSB (trypticase soy broth) culture of Staph epidermidis
a TSA plate culture of E. coli
a culture of E. coli and Staphylococcus mixed together
sterile media: (per person)
3 TSB
2 TSA slants
2 TSA plates
Although your instructor will show you how to perform these procedures,
here are the methods you will use.
ASEPTIC TECHNIQUE:
1. Have both the culture that you are taking the inoculum
from and the new, sterile medium in front of you. Be sure
that the new medium is already labeled so you do not
confuse the various cultures.
2. Pick up both tubes in the hand not using the inoculation
instrument.
3. Heat the inoculating wire of the loop until redhot, and be sure that the ENTIRE wire is sterilized. You are
now ready to pick the inoculum from the bacterial culture.
4. Keeping the sterile inoculation instrument in your hand,
remove both tube caps with your little finger.
5. Run the tops of the tubes through the heat to create an
updraft (taking air contaminants AWAY from the tube entrance).
6. Go into the tube to take your inoculum and QUICKLY place the inoculum into the new
medium tube.
7. Sterilize the tops of the tubes again (to eliminate potential air contamination again) and
replace the caps.
8. Incubate the plates and tubes in the 30 degree C incubator. Look at the section below in
INTERPRETION to read your tube results.
If your goal is to identify the type of growth pattern, then just bring the loop
straight up the slant.
If your goal is to have a luxuriant culture, inoculate in a zig-zag pattern, starting
at the bottom of the slant. This increases the surface area of the culture.
1. Pick up a loopful of your inoculum from either a broth or an agar culture. Using a sterile
agar medium plate (lift the lid just enough to insert the loop), streak a vertical line straight
down.
2. When streaking the agar, keep the loop horizontal and only streak the surface of the agar:
DO NOT DIG INTO THE AGAR.
3. Move the loop in a zig-zag pattern across the agar until 1/3 of the plate is covered, finishing
the first section.
4. Sterilize the loop in the flame and let it cool before continuing to spread the bacteria. You
can do this by 1) sticking the hot loop in the agar at the edge of the agar away from the
bacteria, or 2) just holding the loop for a few seconds while it cools.
5. Rotate the plate about 90 degrees and spread the bacteria from the first streak into a
second area using the same zig-zag spread technique.
6. Sterilize the loop again. Rotate the plate about 90 degrees and spread the bacteria from
the second streak into the 3rd area in the same pattern.
7. Sterilize the loop again. Replace the lid and invert the plate. Incubate the plate.
INTERPRETATION OF RESULTS:
AFTER INCUBATION, check the growth patterns of all tubes and plates.
QUESTIONS:
1. Why streak from the bottom of the agar slant medium up to the top in a straight line, rather
than a back and forth wavy inoculation from side-to-side on the slant?
2. Of what use is it to know what kind of growth pattern on an agar slant or a broth medium
an organism has?
3. Why do you cross over back the 2nd streak section back into the 1st section, and from the
3rd section back across the 2nd section?
4. Why use a streak plate to grow a bacterium rather than an agar medium slant or a broth
medium?